Compacted nucleic acids and their delivery to cells

ABSTRACT

Nucleic acids are compacted, substantially without aggregation, to facilitate their uptake by target cells of an organism to which the compacted material is administered. The nucleic acids may achieve a clinical effect as a result of gene expression, hybridization to endogenous nucleic acids whose expression is undesired, or site-specific integration so that a target gene is replaced, modified or deleted. The targeting may be enhanced by means of a target cell-binding moiety. The nucleic acid is preferably compacted to a condensed state.

This application, which is a national stage application filed under 35USC 371, (PCT/-U.S. Ser. No. 95/03677 which is a continuation-in-part ofSer. No. 08/216,534, filed Mar. 23, 1994, now abandoned, herebyincorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to the compaction of nucleic acids and thedelivery of compacted exogenous nucleic acids to cells of multicellularorganisms, in vivo.

2. Description of the Background Art

Functional exogenous genes can be introduced to mammalian cells in vitroby a variety of physical methods, including transfection, directmicroinjection, electroporation, and coprecipitation with calciumphosphate. Most of these techniques, however, are impractical fordelivering genes to cells within intact animals.

Receptor-Mediated Uncompacted DNA Delivery In Vivo. Receptor-mediatedgene transfer has been shown to be successful in introducing transgenesinto suitable recipient cells, both in vitro and in vivo. This procedureinvolves linking the DNA to a polycationic protein (usuallypoly-L-lysine) containing a covalently attached ligand, which isselected to target a specific receptor on the surface of the tissue ofinterest. The gene is taken up by the tissue, transported to the nucleusof the cell and expressed for varying times. The overall level ofexpression of the transgene in the target tissue is dependent on severalfactors: the stability of the DNA-carrier complex, the presence andnumber of specific receptors on the surface of the targeted cell, thereceptor-carrier ligand interaction, endocytosis and transport of thecomplex to the nucleus, and the efficiency of gene transcription in thenuclei of the target cells.

Wu, et al., U.S. Pat. No. 5,166,320, discloses tissue-specific deliveryof DNA using a conjugate of a polynucleic acid binding agent (such aspolylysine, polyarginine, polyornithine, histone, avidin, or protamine)and a tissue receptor-specific protein ligand. For targeting livercells, Wu suggests "asialoglycoprotein (galactose-terminal) ligands".These may be formed, Wu says, either by desialation of appropriateglycoproteins, or by coupling lactose to non-galactose bearing proteins.The molar ratio of polynucleic acid to conjugate is in the range 1:10 to10:1, more typically 1:5 to 5:1, more preferably 1:2 to 3:1. While notstated by Wu et al., in our hands, Wu's method resulted in structureswith a diameter of at least 80 nm.

Low, et al., U.S. Pat. No. 5,108,921, disclose binding biotin to DNA totransform a cell using receptor mediated endocytosis.

Stomp, et al., U.S. Pat. No. 5,122,466 and McCabe, et al., U.S. Pat. No.5,120,657 disclose attaching DNA to a metal pellet by covalentlyattaching polylysine to the material and then allowing DNA to becomplexed to it. The resulting product is then used for ballistictransformation of a cell. See Stomp, et al., column 7, lines 29-37andMcCabe, et al., column 7, lines 49-65.

Wagner, et al., Proc. Natl. Acad. Sci., 88:4255-4259 (1991) disclosecomplexing a transferrin-polylysine conjugate with DNA for deliveringDNA to cells via receptor mediated endocytosis. Wagner, et al., teachthat it is important that there be sufficient polycation in the mixtureto ensure compaction of plasmid DNA into toroidal structures of 80-100nm diameter, which, they speculate, facilitate the endocytic event.Wagner et al. do not recognize the value of attaining smaller diameterstructures or teach how to obtain a greater degree of compaction. It isbelieved that Wagner et al's structures are multimolecular complexes,which have the disadvantage that they are more vulnerable to macrophagephagocytosis and less amenable to uptake by target tissues.

Direct injection of Naked, Uncompacted DNA. The possibility of detectinggene expression by directly injecting naked DNA into animal tissues wasdemonstrated first by Dubenski et al, Proc. Nat. Acad. Sci. USA,81:7529-33 (1984), who showed that viral or plasmid DNA injected intothe liver or spleen of mice was expressed at detectable levels. The DNAwas precipitated using calcium phosphate and injected together withhyaluronidase and collagenase. The transfected gene was shown toreplicate in the liver of the host animal. Benvenisty and Reshef, Proc.Nat. Acad. Sci. USA, 83:9551-55 (1986) injected calcium phosphateprecipitated DNA intraperitoneally into newborn rats and noted geneexpression in the livers of the animals 48 hr. after transfection. In1990, Wolff et al, Science, 247:1456-68 (1990), reported that the directinjection of DNA or RNA expression vectors into the muscle of miceresulted in the detectable expression of the genes for periods for up to2 months. This technique has been extended by Acsadi et al, NewBiologist, 3:71-81 (1991) to include direct injection of naked DNA intorat hearts; the injected genes were expressed in the heart of theanimals for up to 25 days. Other genes, including the gene fordystrophin have been injected into the muscle of mice using thistechnique. This procedure forms the base of a broad approach for thegeneration of immune response in an animal by the administration of agene by direct injection into the target tissue. The gene is transientlyexpressed, producing a specific antigen (see Donnelly et al, TheImmunologist, 21, pp. 20-26 (1994) for a recent review). However, theDNA used in these experiments has not been modified or compacted toimprove its survival in the cell, its uptake into the nucleus or itsrate of transcription in the nucleus of the target cells.

Behavior of DNA in Solution. DNA is a rod-like molecule in solution, dueto the highly negatively charged nature of its phosphate backbone, andits basic structure can be perturbed by modification of the hydrationshell associated with the helix. This perturbation can be brought aboutin two ways; first, a change in the degree of charge neutralization ofthe DNA molecules resulting in extensive compaction and eventually inthe separation of the DNA phase (precipitation) in the form of compactstructures, and second, a change in the dielectric constant of the DNAhelix leading to the formation of compact structures. Theseperturbations result in a change in the conformation of the DNA moleculepermitting the flexible polymer to bend and become compacted, markedlyaltering the hydrodynamic properties of the DNA molecule. The resultantstructures are thought to be of similar nature to that which the DNAassumes in the chromosomes of higher eukaryotes and inside viralcapsids.

DNA in the nucleus of a higher eukaryote is intimately associated withbasic nuclear proteins (i.e. the histones and protamines) with a highcontent in lysine and arginine (histones) or arginine (protamines). Thecomplex of DNA with these basic proteins is responsible for the controlof DNA compaction that occurs upon chromosome formation and is thoughtto play a role in the regulation of gene expression. DNA compaction,which occurs physiologically in viruses, bacteria and eukaryote nuclei,has been extremely difficult to reproduce in the laboratory.Theoretically, due to the highly negatively charged nature of the DNAbackbone, a change in the degree of charge neutralization of the DNAresults in extensive compaction and eventually in the separation of theDNA phase (precipitation) in the form of compact structures. However,the behavior of DNA-polycation complexes in solution is dependent on themethod for complexing DNA with the poly caionic protein.

Studies by Olins, Olins and von Hipple (J. Mol. Bio. 24, 157-176, 1967)using cationic homopolypeptides as models for nucleoprotein complexformation presented evidence for the formation of specific complexes ofDNA with cationic polypeptides (poly-L-lysine, poly-L-arginine andpoly-L-ornithine) after "annealing" of both components in solution. Thisprocedure involved step-down dialysis from NaCl concentrations of 2M to0.010M.

Several comments may be made on this study. First, thermal denaturationof complexes formed by the addition of polycation to DNA establishedthat polycation binding to DNA occurred in every case studied, andresulted in the stabilization of the double stranded structure of DNA.It is important to note that this system differs from that in which achange in the dielectric constant (i.e. alcohol dehydration) results inDNA collapse with no change in the thermal denaturation characteristicsof the DNA. Second, spectrophotometric studies indicated that theabsorbance maxima at 260 nm was shifted slightly to the red with aprogressive increase in turbidity at wavelengths greater than 300 nm (aregion in which neither the polycation nor the DNA show any absorbance).These characteristics were thought to indicate that a smallconformational change, occurring possibly through the interactionbetween DNA and the polypeptide, was being detected by an anomalousabsorption spectra. Third, the complexes formed by the addition of basicpolypeptides to DNA resulted in molecular aggregation and the formationof precipitates.

Optical Rotatory Dispersion and Circular Dichroism were applied to thestudy of the interaction between basic homopolypeptides and DNA insolution. Shapiro, Leng and Felsenfeld (Biochemistry, 8:3219-3232, 1969)elucidated the changes in secondary structure associated with theformation of DNA complexes by examining their optical rotation, using aprotocol for complexing polylysine to DNA essentially different to thatof annealing both components in a step-down salt dialysis. they directlymixed polylysine and DNA in a high salt solvent (1M NaCl), whichresulted in the formation of "soluble" complex. A high degree ofturbidity is associated with the complex in solution, indicatingaggregation of the components. Aggregation was occurring in the samplesused to determine the optical rotatory properties of the complex sincethe circular dichroism spectra approached the baseline asymptotically atwavelengths in the range of 320 to 360 nm. The anomalous spectrum wasalways associated with turbidity. We have inferred that the opticalactivity changes arose from the formation of higher order molecularcomplexes upon aggregation.

DNA complexes obtained under the experimental conditions described abovehave a median sedimentation coefficient varying between about 5000 and10000 units. The average particle had a diameter of 340 nm, (calculatedusing information provided by light scattering) and the particles had anaverage dry mass corresponding to about 70 nucleic acid/polypeptidemolecular units. The information provided by these studies, while notabsolutely quantitative, delineates the structural changes that DNAundergoes after binding to a basic polypeptide.

Several aspects of the structure of DNA-polybase complexes in solutionhave been investigated (Haynes, Garrett and Gratzer, Biochemistry,9:4410-4416, 1970). Electron microscopy confirmed the ordered nature ofthe complexes described by Shapiro et al; DNA structures formed asdoughnut-shaped toroids, with an external diameter of 300 nm. The C. D.and electron microscopic features of DNA-poly-base complexes correspondto structural factors residing in the Watson-Crick DNA helix, sincesingle-stranded polynucleotides-polybase complexes i.e. rRNA, po.y(A),poly (U), etc.) do not show anomalous optical activity. Also, orderedstructures can be detected in the electron microscope. In order toclarify whether a change in base tilt and/or helix pitch could beobserved in the complexes, the X-ray diffraction pattern of thecomplexes was determined. The double helix is in the normal B formobtained for free DNA in aqueous solutions; no obvious transitions werefound to the C or A forms of DNA, suggesting the existence of adifferent structural form when the DNA is complexed to basicpolypeptides in solution. There is also an association of DNA-polybasecomplexes which involves direct pairing of charges, as shown by theprogressive displacement of counter-ions in DNA-polylysine complexes asthe salt concentration is decreased. Any strong interaction of thecharged amino group with a base is therefore very improbable. Thus, theanomalous rotatory strength of DNA in solution arises from chiralpacking, the kind of phenomenon associated with the appearance of alarge periodicity in the asymmetric packing of molecules in the sameplane.

Lerman et al., Proc. Nat. Acad. Sci. USA, 68:1986-90 (1971) report thatwhen a dilute solution of phage DNA is mixed with a sufficiently highconcentration of a simple neutral polymer (polyethylene oxide) in thepresence of high NaCl (a simulated intracellular environment), the phageDNA molecules collapse into particles approaching the compactness of thecontents of phage heads. The structure of DNA complexes was. resolved byGosule and Schellman (Nature 259: 333-335, 1976). Their publicationalong with a more detailed report (Gosule, L., Chattoraj. D. K., andSchellman. J., Advances in Polyamine Research 1: 201-215, 1978), showedthat the compaction of DNA (in a very dilute solution) by basicpolypeptides (spermine and spermidine), under the conditions firstdescribed by Li, Biopolymers, 12:287 (1973), resulted in toroidstructures. The complexes generated by Gosule and Schellman had anunimolecular structure consisting of a single DNA unit of phage DNAcompacted to a maximum radius of about 50 nm. The authors note thatpolyamines are known to exist in bacterial cells. DNA compaction is alsodiscussed by Laemmli, PNAS 72:4288-92 (1975) and Post and Zimm,Biopolymers, 21:2123-32 (1982).

All references cited in this specification are hereby incorporated byreference. No admission is made that any reference constitutes priorart. The discussion of the references states what their authors assertand applicants reserve the right to challenge the accuracy andpertinency of the cited documents.

SUMMARY OF THE INVENTION

The present invention relates to a method for compacting nucleic acids,substantially without aggregation, and to therapeutic use of thecompacted DNA. The compacted nucleic acid can be efficiently deliveredacross the membrane of a living cell, especially a cell in amulticellular organism. DNA condensed into small particles may be moresuitable for nuclear translocation through the nuclear pores and may beprotected against nucleases. When the nucleic acid includes anexpressible gene, that gene can be expressed in the cell.

In some embodiments, a tissue-specific carrier molecule is prepared,which is a bifunctional molecule having a nucleic acid-binding moietyand a target tissue-binding moiety. The nucleic acid is then compactedat high concentrations with the carrier molecule at a critical saltconcentration. The nucleic acid-loaded carrier molecule is thenadministered to the organism. Each carrier molecule bears a singlenucleic acid molecule.

In other embodiments, a target tissue-binding carrier molecule is notused. However, the nucleic acid is still compacted by complexing with acarrier molecule comprising a nucleic acid binding moiety which reducesinteractions between the nucleic acid and the solvent. The compactedcomplexes are administered to the organism.

The appended claims are hereby incorporated by reference into thisdescription as a recitation of preferred embodiments of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1J - Physical characterization of thegalactose-poly-L-lysine/DNA complexes.

FIG. 1A shows CD spectra associated with normal DNA in solution and withcertain poly-L-lysine/DNA complexes. Sixty micro grams of RNA-freeCMV-β-galactosidase plasmid (dissolved in TE buffer, pH 8), 150 μl of700 mM NaCl were vortexed at medium speed in a VIBRAX apparatus(IKA-VIBRAX-VXR). Nineteen micrograms of α-galactopyranosyl-phenylisothiocyanate/poly-L-lysine biconjugate in 150 μl of 700 mM NaCl wereadded dropwise to the vortexing solution of DNA. The slow addition ofthe polycation results in the formation of a turbid solution which isdissolved by the slow, stepwise addition of 3 μl aliquots of 5M NaCl.The disappearance of the turbidity was monitored by eye and thesolutions of DNA/poly-L-lysine complexes were investigated by CD. Atthis point (0.97M NaCl), the CD spectrum was that characteristic ofaggregated DNA. Further addition of 2 μl aliquots of 5M NaCl (resultingin a concentration of 1.031M NaCl) yielded the CD spectrum expected fora condensed (or a relaxed) DNA complex. The CD spectrum of uncomplexeddouble stranded DNA at 1M NaCl was also taken. The spectra were obtainedusing a JASCO-600 spectropolarimeter with a 0.1 cm cuvette. The spectrumof the buffer was subtracted in each case.

FIGS. 1B-1G are electronic micrographs (EM). 1B-1D, 1F and 1G are takenat 300,000×. The bar in 1D represents 33.3 nm. FIG. 1E was taken at600,000×, and the bar is 16.6 nm long. Uranyl acetate staining wasperformed as previously described. (Ennever, et al., Biochem. Biophys.Acta, 826:67 (1985)). Briefly, the grid was subjected to glow dischargeprior to staining. A drop of DNA solution was added to the grid, blottedand stained using 0.04% uranyl acetate.

For the EM studies shown in FIGS. 1B-1F, 60 μg of PEPCK-hFIX plasmid DNA(dissolved in TE buffer, pH 8), in 150 μl of 700 mM NaCl were vortexedat medium speed in a VIBRAX apparatus (IKA-VIBRAX-VXR). Nineteenmicrograms of α-galactopyranosyl-phenyl isothiocyanate/poly-L-lysinebioconjugate in 150 μl of 700 mM NaCl were added dropwise to thevortexing solution of DNA. The slow addition of the polycation resultsin the formation of a turbid solution which is dissolved by the slow,stepwise addition of 3 μl aliquots of 5M NaCl. The disappearance of theturbidity was monitored by eye and the solution of DNA/poly-L-lysinecomplexes was investigated by EM (FIG. 1C). Further addition of 2 μlaliquots of 5M NaCl resulted in structural changes as shown in FIGS. 1Dand 1E.

FIG. 1B is an EM of uncomplexed DNA (1 ug/ml at 1M NaCl). FIG. 1Cdepicts a DNA complex at a suboptimal concentration of NaCl (760 mM).The DNA is in the aggregated state; clusters of unimolecular toroids arevisible. In FIG. 1D the DNA complex is at an optimal concentration ofNaCl for the complex in question (968 mM). The DNA is properlycondensed; only individual toroids can be seen. For FIG. 1E, fourcomplexes of DNA from FIG. 1D were selected and printed at highermagnification. In FIG. 1F, we see a DNA complex, at a concentration of1.068M NaCl, which is above optimal for condensation of this complex.The DNA is in the relaxed state. Note the branched unimolecular toroidsin which a nucleus of condensation is visible and the rod-like DNAfibers.

Differences in concentration of NaCl required for aggregated, condensed,and relaxed states in the above experiments represent DNA or polycationspecific differences.

In a third experiment, complexes of CMV-β-galactosidase andgalactosylated poly-L-lysine were formed essentially as in Wu et al.Briefly, plasmid DNA and galactosylated poly-L-lysine were combined in3M NaCl. The samples were incubated for 1 hour at room temperature, thendialyzed against 0.15M NaCl for 16 hr through membranes with a3,500-dalton molecular mass limit. On visual inspection, no precipitateswere present in the dialysate.

FIG. 1G is an electron micrograph of the resulting DNA complex, which isin the multimolecular aggregated state. Note that the toroids here arelarger than in 1C or 1D (the scale is the same). FIG. 1H shows the CDspectrum from 240 to 300 nm for uncomplexed DNA and for aggregatedmultimolecular DNA/poly-L-Lys complexes, so as to highlight theinversion of the normal DNA spectrum maximum at 269 nm. This inversionis characteristic of multimolecular aggregation.

In another experiment, sixty micrograms of PEPCK-hFIX plasmid DNA(dissolved in TE buffer, pH 8), in 150 μl of 200 mM NaCl were vortexedat medium speed in a VIBRAX apparatus (IKA-VIBRAX-VXR). Nineteenmicrograms of α-galactopyranosyl-phenyl isothiocyanate/poly-L-lysinebiconjugate in 150 μl of 200 mM NaCl were added dropwise to thevortexing solution of DNA. The addition of the polycation resulted inthe formation of precipitates on visual inspection.

FIG. 1I is a CD spectrum, given by a precipitated DNA complex. It isessentially flat from 240 to 300 nm. FIG. 1J is an electron micrographof the precipitated DNA.

FIGS. 2A and 2B - Functional relevance and specificity of the genetransfer system. (FIG. 2A) The relative concentration of human factor IXin the blood of animals treated with the DNA complex was evaluated bymeasuring the procoagulant activity of human factor IX. A modificationof the one stage, kaolin-activated, partial thromboplastin time withfactor IX-deficient human plasma was used. Blood samples were obtainedfrom experimental animals by venipuncture. One fiftieth volume of 500 mMsodium citrate, pH 5.0, was added to prevent coagulation, and the plasmawas stored at -20° C. The samples were assayed in duplicate, and theiractivity was compared to the functional activity of pooled plasma from24 normal adult human males. In all calculations, one unit of factor IXactivity in one ml of normal human plasma is equivalent to 100%functional activity or approximately 3 μg of factor IX per ml.Background human factor IX activity in the rat plasma was subtractedprior to graphic representation. (FIG. 2B) Transfected animals were feda carbohydrate-free/high protein diet for one week. Blood samples weretaken at the initiation of the treatment and after one week on the dietand analyzed by Western blot hybridization. The animals at 8 and 12 dayswere compared with transfected rats fed a standard chow diet. The datawere obtained by densitometric analysis of Western blot photographicfilms and indicate fold increase in human factor IX protein after thedietary treatment.

FIG. 3.- Tissue specificity of mannosylated DNA complex in targeting DNAto the macrophages in vivo. Mannosylated poly-L-lysine was conjugated toSV40/luciferase DNA. 300 μg of the DNA complex were introduced into thecaudal vena cava of rats. Four days after injection tissue extracts weremade and assayed for luciferase activity. The luciferase activity isplotted as Integrated Light Units per milligram of protein extract fromspleen, liver and lung. In other tissues no activity was found. Data areexpressed as means±standard error of the mean (SEM). The light bars arethe non-transfected controls (n=4), and the dark bars, animalstransfected with mannosylated poly-L-lysine/DNA complexes (n=5).

FIG. 4.- Specificity of mannosylated DNA complex in targeting DNA toprimary culture of macrophages in vitro. Primary cultures of peritonealmacrophages were transfected with either galactosylated poly-L-lysine(light bars) or mannosylated poly-L-lysine (dark bars) conjugated toa-SV40/luciferase DNA. At the indicated times (2, 4, 8, and 24 hours)cells were washed. Twenty-four hours after transfection, cells wereharvested and assayed for luciferase activity. The luciferase activityis plotted as Relative Luciferase Activity after being standardized bythe activity found in untransfected controls. Data are expressed asmeans±standard error of the mean (SEM).

FIG. 5.- Competition between the mannosylated DNA complex andmannosylated bovine serum albumin for binding to the Mannose receptor ofmacrophages. Primary culture of peritoneal macrophages were transfectedwith mannosylated poly-L-lysine conjugated to SV40/luciferase DNA (T).Prior to the addition of the DNA complex a 100-fold excess mannosylatedbovine serum albumin was added to one set of plates (Tc).Non-transfected controls (NT) were also assayed for luciferase activity24 hours after transfection. The luciferase activity is plotted asRelative Luciferase Activity after being standardized relative to theactivity found in untransfected controls. Data are expressed asmeans±standard error of the mean (SEM).

FIG. 6.- In vivo gene transfer using the anti-rat plg-RFab-poly-L-lysine conjugated DNA complex. Fab-poly-L-lysine wasconjugated to SV40/luciferase DNA and introduced into the caudal venacava of rats (Transfected) (n=3). Untransfected controls (Control)(n=3), animals injected with an Fab-poly-L-lysine-DNA complex containingan Fab fragment obtained from an irrelevant IgF (IFab) (n=3), andanimals injected with a DNA complex that does not contain anSV40/Luciferase gene (IDNA) (n=3), were run as controls. Two days afterinjection tissue extracts were prepared and assayed for luciferaseactivity. The luciferase activity is plotted as Integrated Light Unitsper milligram of protein extract. Data are expressed as means±standarderror of the mean (SEM).

FIG. 7.- Time-course of expression in lung and liver of animals injectedusing the anti-rat plg-R Fab-poly-L-lysine conjugated DNA complex.Fab-poly-L-lysine was conjugated to SV40/luciferase DNA and introducedinto the caudal vena cava of rats (n=9). Rats were killed 2 (n=3), 4(n=3) and 6(n=3) days after injection. Lung and liver extracts wereprepared and assayed for luciferase activity. The luciferase activity isplotted as Integrated Light Units per milligram of protein extract usinga logarithmic scale. Data are expressed as means±standard error of themean (SEM).

FIG. 8.- Competition between the galactoslyated DNA complex andasialoorosomucoid for binding to the ASGP receptor of HepG2 cells. HepG2hepatoma cells were transfected with galactosylated poly-L-lysineconjugated to PEPCK-hFIX DNA. Prior to the addition of the DNA complex a100-fold excess asialoorosomucoid was added to one set of plates (+Comp.). DNA internalization was monitored by slot-blot hybridization ofthe culture medium containing the DNA complex. Data are expressed aspercentage of DNA internalized by the receptor at different times aftertransfection.

FIG. 9 Direct injection to the muscle and liver of naked DNA vs.condensed DNA. One hundred micrograms of naked DNA encodingSV40-luciferase were injected into the liver and abdominal muscle of tworats. The same amount of the pSV40-luciferase plasmid complexed topoly-L-lysine and condensed as described in Example 1 was injected aswell into the liver and abdominal muscle of another two animals. Ratswere sacrificed 48 hours post-injection. A piece of liver and abdominalmuscle were homogenized in lysis buffer and cell lysates were analyzedfor luciferase activity. All luciferase measurements were performed intriplicate, expressed as an average of the values and standardized fortotal protein. FIG. 9 shows the integrated luciferase units per mg ofprotein in the two different sets of animals.

FIG. 10 Direct injection into the brain tectum of naked DNA vs.condensed DNA. Intratectal injections of naked and poly-L-lysinecondensed plasmid DNA can achieve high levels of expression in the cellbody of the neuron over 20 days. β-galactosidase activity in retinasfrom rats whose brains were injected into the tectal areas andadministered with either naked pCMV-lacZ, or condensed pCMV-lacZ(pCMV-lacZ+lys) at the concentrations shown. When the DNA is notcondensed with poly-L-lysine the level of expression returns tobackground after 10 days post-injection.

FIG. 11. Changes in the absorbance of the DNA complexes during thecondensation process. A plasmid containing the chimeric CMV-hLDLreceptor gene was condensed with poly-L-lysine, using the proceduredescribed in detail in Example 1. After the addition of poly-L-lysinethe absorbance of the solution at 260 nm was determined. ConcentratedNaCl was then added stepwise and the absorbance determined. The expectedabsorbance for the DNA contained in the complex is indicated by thedotted line. The initial NaCl concentration used in the condensationreaction was 500 mM.

FIG. 12 Relationship between the structure of the DNA complex and itsfunction in adult rats. DNA-galatosylated poly-lysine complexes wereprepared which correspond to various states of condensation/aggregationshown in FIG. 1B-1G. The DNA consisted of the SV40 promoter linked tothe structural gene for P. pyralis luciferase gene. Rats were injectedin the caudal vena cava with 300 μg of the various DNA complexes and theactivity of luciferase was determined in extracts from the liver and thespleen 48 hr after injection. Each bar represents the mean±SEM for threerats; control rats were not injected with the DNA complex.

FIG. 13 Introduction of 3 mg of PEPCK-hLDLr in its relaxed (noncomplexed) vs. condensed form. In order to introduce the DNA complexinto the animal, we perform a single injection of 3-10 ml of theDNA-complex solution (.sup.˜ 400-900 mM NaCl) into the marginal ear veinof the rabbit. Approximately 1.5 ml of blood was drawn at the timesindicated from the ear artery at 4 p.m. The determination of theconcentration of serum cholesterol was performed in the ClinicalLaboratory of University Hospitals of Cleveland from 300 μl of serum.The administration of a DNA complex solution containing 3 mg of thepPEPCK-hLDLR plasmid in a relaxed state to rabbit #676 did not result ina significant decrease (first arrow) in total serum cholesterol levels.A second injection of DNA complexes appropriately condensed containing 3mg of the same DNA (second arrow) caused a 20% reduction of the levelsof cholesterol in the blood. Four weeks after this secondadministration, cholesterol returned to approximately pre-treatmentlevels, reaching a peak at about 35 days.

FIG. 14 Injection of the poly-L-lysine/DNA complex containing 9 mg ofthe chimeric PEPCK-hLDLr gene. In our second experiment, 9 mg of thePEPCK-hLDLr gene appropriately condensed with galactosylatedpoly-L-lysine were administered to rabbit #737. As shown in FIG. 14, thetreatment resulted in a 38% reduction of total serum cholesterol levelswhich lasted for about 5 weeks. Thus, a 3-fold increase in the dose ofDNA complex resulted in a 2-fold reduction in total serum cholesterollevels.

FIG. 15 Injection of the poly-L-lysine/DNA complex containing 3 mg ofthe chimeric CMV-hLDLr gene. The administration of a DNA complexsolution containing 3 mg of the chimeric CMV-hLDL receptor gene torabbit #16 resulted in a maximal reduction of 30% in total serumcholesterol levels (FIG. 15). Eleven weeks after the injection,cholesterol levels are still 20% below those observed before thetreatment.

FIGS. 16A and 16B Injection of multiple doses of the poly-L-lysine/DNAcomplex containing 3 mg of the chimeric CMV-hLDLr gene. Rabbits #775(FIG. 16A) and #774 (FIG. 16B) were injected with 3mg of the pCMV-hLDLRcomplex. In rabbit #775, this caused a maximal 24% reduction incholesterol concentration in the blood, 3 weeks after treatment. Twoadditional injections did not result in a further significant reductionin serum cholesterol. In Rabbit #774, we observed a 36% decrease in thecholesterol levels in the blood (FIG. 16B) after the initial injeciton.Four reinjections once every 2 weeks were performed with the same amountof DNA complex. Two of them resulted in a minimal effect while the othertwo in a null reduction of total serum cholesterol levels. However,after five administrations of the DNA complex solution containing 3 mgof pCMNV-hLDLr, the concentration of cholesterol had dropped about 48%with respect to pre-treatment levels.

Rabbit #774 was then treated with 10 mg of lovastatin (striped bar) perday for 10 weeks. A further 20% reduction in the levels of cholesterolhas been observed. mhe inhibition of the endogenous pathway forcholesterol synthesis has thus brought the cholesterol concentration ofrabbit #774 to 40% of that prior to the first gene transfer (FIG. 16B).

FIG. 17 Mock-injection of the poly-L-lysine/DNA complex containing 3 mgof the chimeric SV40-luciferase gene (irrelevant DNA). In order tocontrol for a possible nonspecific reduction in total serum cholesterollevels by injecting rabbits with the galactosylated poly-L-lysine/DNAcomplexes in a solution with high NaCl concentration (.sup.˜ 900 mM), wehave administered a DNA complex solution containing an irrelevant DNAsuch as the luciferase gene into rabbit #775. FIG. 17 shows that theinjection results in a non-significant (≦12%) and transient (≦5 days)reduction in the serum cholesterol concentration. Thus, we haveconfirmed that the reduction in total serum cholesterol levels after theinjection of appropriately condensed DNA particles encoding the humanLDL receptor gene are not a result of either the high NaCl concentrationof the solution or the presence of galactosylated poly-L-lysine/DNAparticles.

FIG. 18 Relationship of turbidity to NaCl concentration. The figureshows the effect of initial and current NaCl concentration on theturbidity of a DNA/poly-lysine solution. Each line represents adifferent initial concentration.

FIG. 19 Effect of poly-L-lysine length on condensation concentration ofNaCl.

FIGS. 20A-20E CD spectra for different complexes. CD spectra were takenin a 0.1 cm path-length cuvette. The DNA was complexed withpoly-L-lysine at identical molar ratios of amino to phosphate groups andvarious CD spectra compared: (FIG. 20A) standard control for DNA in 1MNaCl; (FIG. 20B) Ψ-DNA as observed at a concentration of NaCl at whichmultimolecular aggregation occurs; (FIG. 20C) aggregated DNA showsturbidity and decreased ellipticity; (FIG. 20D) condensed, unimolecularcomplexes of DNA; (FIG. 20E). relaxed DNA complex spectrum. The spectawas taken at equal concentrations of polymer and the signal for thebuffer was subtracted in each case. details of the assay are presentedin the Methods.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The Multicellular Organism

Any multicellular organism into which it may be desirable to introduceexogenous nucleic acid is a potential subject for the present invention.The multicellular organism may be a plant or an animal, preferably thelatter. The animal is preferably a vertebrate animal, and morepreferably a higher vertebrate, i.e., a mammal or bird, the former beingespecially preferred. Among mammals, preferred subjects are human andother primates, laboratory animals such as mice, rats, rabbits andhamsters, pet animals such as dogs and cats, and farm animals such ashorses, cows, goats, pigs and sheep. It will be noted that these animalscome from four orders of class Mammalia: Primata, Rodenta, Carnivora andArtiodactyla.

The Target Cell

The target cells may belong to tissues (including organs) of theorganism, including cells belonging to (in the case of an animal) itsnervous system (e.g., the brain, spinal cord and peripheral nervouscells), the circulatory system (e.g., the heart, vascular tissue and redand white blood cells), the digestive system (e.g., the stomach andintestines), the respiratory system (e.g., the nose and the lungs), thereproductive system, the endocrine system (the liver, spleen, thyroids,parathyroids), the skin, the muscles, or the connective tissue.

Alternatively, the cells may be cancer cells derived from any organ ortissue of the target organism, or cells of a parasite or pathogeninfecting the organism, or virally infected cells of the organism.

A useful procedure for hepatic gene therapy requires an efficient andrelatively non-invasive approach to the introduction of genes ofinterest into the liver. Several techniques, employing receptor mediatedgene transfer, have been used with some success. However, there is aneed for a readily reproducible procedure which results in the prolongedexpression of the transgene in the liver, even in the absence of partialhepatectomy, and which therefore could be used for human gene therapy.Exogenous DNA has been introduced into hepatocytes of adult animals bytargeting the asialoglycoprotein (ASGP) receptor by means of aligand-poly-L-lysine biconjugate. For the ligand-targeting technique tobe efficient, the DNA must be in a form which permits it to remainintact in the blood and is small enough to be recognized by the ASGPreceptor on the surface of the hepatocytes. Wagner, et al. (1991) havetargeted genes to the transferrin receptor in hepatoma cells bycondensing the DNA with a poly-L-lysine/transferrin conjugate, into acomplex with a diameter of 80-100 nm. This size DNA conjugate isappropriate for recognition by the transferrin receptor in hepatomacells, but the ASGP receptor of hepatocytes discriminates againstligands larger than 10-20 nm in diameter.

We have developed a procedure for the introduction of genes into theliver of adult animals by receptor mediated uptake which resulted in theexpression of the gene for 140 days (the duration of the experiment).This procedure has potential for application to human gene therapy. Themajor advantages of this method are (i) the ease of preparation of theDNA complex; (ii) the ability to target genes to specific tissues; (iii)the prolonged expression of the gene in the liver; (iv) the relativesafety of the complex, since it is devoid of infectious viral DNA; and(v) the episomal maintenance of the introduced gene.

Targeting

A. Generally

"Targeting" is the administration of the compacted nucleic acid in sucha manner that it enters the target cells in amounts effective to achievethe clinical purpose. In this regard, it should be noted that DNA andRNA are capable of replication in the nucleus of the target cell, and inconsequence the ultimate level of the nucleic acid in the cell mayincrease after uptake. Moreover, if the clinical effect is mediated by aprotein expressed by the nucleic acid, it should be noted that thenucleic acid acts as a template, and thus high levels of proteinexpression can be achieved even if the number of copies of the nucleicacid in the cell is low. Nonetheless, it is desirable to compact highconcentrations of DNA to increase the number of target cells which takeup the DNA and the number of DNA molecules taken up by each cell.

The route and site of administration may be chosen to enhance targeting.For example, to target muscle cells, intramuscular injection into themuscles of interest would be a logical choice. Lung cells might betargeted by administering the compacted DNA in aerosol form. Thevascular endothelial cells could be targeted by coating a ballooncatheter with the compacted DNA and mechanically introducing the DNA.

In some instances, the nucleic acid binding moiety, which maintains thenucleic acid in the compacted state, may also serve as a targetingagent. Polymers of positively charged amino acids are known to act asnuclear localization signals (NLS) in many nuclear proteins. ApSV40-luciferase DNA condensed with poly-L-lysine, was injected in situinto the abdominal muscle of rats. Despite the absence of an explicittarget cell binding moiety, we observed a 20-fold higher luciferaseactivity in rats injected with the complexed DNA than in the ratinjected with naked DNA. Nonetheless, in some embodiments, targeting maybe improved if a target cell binding moiety is employed.

B. Use of a Target Cell Binding Moiety

If a TBM is used, it must bind specifically to an accessible structure(the "receptor") of the intended target cells. It is not necessary thatit be absolutely specific for those cells, however, it must besufficiently specific for the conjugate to be therapeutically effective.Preferably, its cross-reactivity with other cells is less than 10%, morepreferably less than 5%.

There is no absolute minimum affinity which the TBM must have for anaccessible structure of the target cell, however, the higher theaffinity, the better. Preferably, the affinity is at least 10³liters/mole, more preferably, at least 10⁶ liters/mole.

The TBM may be an antibody (or a specifically binding fragment of anantibody, such as an Fab, Fab, V_(M), V_(L) or CDR) which bindsspecifically to an epitope on the surface of the target cell. Methodsfor raising antibodies against cells, cell membranes, or isolated cellsurface antigens are known in the art:

a. production of immune spleen cells: immunization with soluble antigensHurrell, J. G. R. (1982) Monoclonal Antibodies: Techniques andApplications. CRC Press, Boca Raton, Fla.

b. immunization with complex antigens: membranes, whole cells andmicroorganisms. Hurrell, J. G. R. (1982) Monoclonal Antibodies:Techniques and Applications. CRC Press, Boca Raton, Fla.

c. production of monoclonal supernatants and ascots fluids. Andrew, S.M. and Titus, J. A. (1991). Purification of Immunoglobulin G. in CurrentProtocols in Immunology (J. E. Coligan, A. M. Kruisbeek, D. H. J.Margulies, E. M. Shevach and W. Strober, ed.) pp. A.3.9-A.3.12. GreenePublishing Wiley-Interscience, New York.

d. production of polyclonal antiserum in rabbits. Garvey J. S., Cremer,N. E. and Sussdorf, D. H (eds) (1977) Methods in Immunology: ALaboratory Text for Instruction and Research, Third Edition. W. A.Benjamin, North Hampton, Mass.

e. production of anti-peptide antibodies by chemical coupling ofsynthetic peptides to carrier proteins Jemmerson, R., Morrow, P. I.,Klinman, N. I and Patterson, Y. (1985). Analysis of an evolutionaryconserved site on mammalian cytochrome C using synthetic peptides. Proc.Natl Acad. Sci, U.S.A. 82, 1508-1512.

The TBM may be a lectin, for which there is a cognate carbohydratestructure on the cell surface.

The target binding moiety may be a ligand which is specifically bound bya receptor carried by the target cells.

One class of ligands of interest are carbohydrates, especially mono- andoligosaccharides. Suitable ligands include galactose, lactose andmannose.

Another class of ligands of interest are peptides (which here includesproteins), such as insulin, epidermal growth factor(s), tumor necrosisfactor, prolactin, chorionic gonadotropin, FSH, LH, glucagon,lactoferrin, transferrin, apolipoprotein E, gp120 and albumin.

The following table lists preferred target binding moieties for variousclasses of target cells:

    ______________________________________    Target Cells   Target Binding Moiety    ______________________________________    liver cells    galactose    Kupffer cells  mannose    macrophages    mannose    lung           Fab fragment vs. polymeric                   immunoglobulin receptor (Pig R)    adipose tissue,                   insulin    lymphocytes    Fab fragment vs. CD4 or gp120    enterocyte     Vitamin B12    muscle         insulin    fibroblasts    mannose-6-phosphate    nerve cells    Apolipoprotein E    ______________________________________

Target binding moiety is not strictly necessary in the case of directinjection of the NABM/NA condensed complex. The target cell in this caseis passively accessible to the NABM/NA condensed complex by theinjection of the complex to the vicinity of the target cell.

C. Liposome-Mediated Gene Transfer

The possibility of detecting gene expresson by encapsulating DNA into aliposome (body contained by a lipid bilayer) using various lipid andsolvent conditions, and injecting the liposome into animal tissues, hasbeen extensively demonstrated (1-7). However, despite the potential ofthis technique for a variety of biological systems, the DNA used inthese experiments has not been modified or compacted to improve itssurvival in the cell, its uptake into the nucleus or its rate oftranscription in the nucleus of the target cells. Thus, these procedureshave usually resulted in only transient expression of the gene carriedby the liposome (4,5).

Cationic lipids have been successfully used to transfer DNA. Thecationic component of such lipids can compact DNA in solution (1-3, 7).This method has been shown to result in heavily aggregated DNA complexes(1,2) that, when used for transfecting the DNA in vitro, results inincreased efficiency of gene transfer and expression (relative to nakedDNA). Although the formation of these complexes can promote genetransfer in vitro, the injection of such complexes in vivo does notresult in long lasting and efficient gene transfer. Our condensationprocedure could thus provide structural features to the DNA/cationiclipid complex that will make it more amenable to prolonged in vivoexpression. We believe that the combination of such methods could beaccomplished by either of two procedures:

1. Formation of condensed DNA complex that is later encapsulated usingneutral lipids into liposome bodies, or

2. Using the procedure described in this patent, the formation of highlycondensed unimolecular DNA complexes upon condensation with cationiclipids could be accomplished.

These complexes should provide a higher efficiency of gene transfer intotissues of animals in vivo.

Our procedure for the condensation of DNA, if coupled to theencapsulation of the resulting compacted DNA into a liposome body, couldprovide a variety of advantages for transfection into animals:

1. The liposome promotes the passive fusion with the lipid bilayer ofthe cytoplasmic membrane of mammalian cells in tissues.

2. The condensed DNA could then transfer the genetic information with ahigher efficiency through the cell compartments to the nucleus for itsexpression.

3. Condensed DNA could be protected against degradation inside the cell,thus augmenting the duration of the expression of the newly introducedgene.

4. Possible immunological response to the polycation condensed DNA couldbe avoided by the encapsulation with the immunologically inert lipidbilayer.

References

1 Ghirlando, R., Wachtel, E. J, Arad, T., and Minsky, A. (1992) DNApackaging induced by micellar aggregates: A novel in vitro DNAcondensation system. Biochemistry, 31, 7110-7119.

2 Braulin, W. H., Strick, T. J., and Record, M. T.,Jr. (1982)Biopolymers 21, 1301-1309.

3 Zhu, N., Liggitt, D., Liu, Y., Debs, R. (1993) Systemic geneexpression after intravenous DNA delivery into adult mice. Science, 261,209-211.

4 Alino, S. F., Bobadilla, M., Garcia-Sanz, M., Lejarreta, M., Unda, F.,and Hilario, E. (1993) In vivo delivery of human α1-antitrypsin gene tomouse hepatocytes by liposomes. Biochem. Biophys. ResearchCommunications 192, 174-181.

5 Takeshits, S., Losordo, D. W., Kearney, M., Rossow, S. T., and Isner,J. M. (1994) Time course of recombinant protein secretion afterliposome-mediated gene transfer in a rabbit arterial organ culturemodel. Lab. Invest. 71, 387-391.

6 Jarnagin, W. R., Debs, R. J., Wang, S. S., and Bissell, D. M. (1992)Nucleic Acids Res. 20, 4205-4211.

7 Philip, R., Liggitt, D., Phillip, M., Dazin, P., Debs, R. (1993) Invivo gene delivery. Efficient transfection of T lymphocytes in adultmice. J. Biol. Chem. 268, 16087-16090.

The Nucleic Acid Binding Moiety

Any substance which binds reversibly to a nucleic acid may serve as thenucleic acid binding moiety (NABM), provided that (1) it bindssufficiently strongly and specifically to the nucleic acid to retain ituntil the conjugate reaches and enters the target cell, and does not,through its binding, substantially damage or alter the nucleic acid and(2) it reduces the interactions between the nucleic acid and thesolvent, and thereby permits condensation to occur. The ultimatecriterion is one of therapeutic effectiveness of the conjugate.

Preferably, the NABM is a polycation. Its positively charged groups bindlonically to the negatively charged DNA, and the resulting chargeneutralization reduces DNA-solvent interactions. A preferred polycationis polylysine. Other potential nucleic acid binding moieties includeArg-Lys mixed polymers, polyarginine, polyornithine, histones, avidin,and protamines.

The Nucleic Acid

Basic procedures for constructing recombinant DNA and RNA molecules inaccordance with the present invention are disclosed by Sambrook, J. etal., In: Molecular Cloning: A Laboratory Manual, Second Edition, ColdSpring Harbor Press, Cold Spring Harbor, N.Y. (1989), which reference isherein incorporated by reference.

The nucleic acid may be a DNA, RNA, or a DNA or RNA derivative such as aderivative resistant to degradation in vivo, as discussed below. Withinthis specification, references to DNA apply, mutatis mutandis, to othernucleic acids as well, unless clearly forbidden by the context. Thenucleic acid may be single or double stranded. It is preferably of 10 to1,000,000 bases (or base pairs), more preferably 100 to 100,000, and thebases may be same or different. The bases may be the "normal" basesadenine (A), guanine (G), thymidine (T), cytosine (C) and uracil (U), orabnormal bases such as those listed in 37 CFR § 1.822 (p) (1). Thenucleic acid may be prepared by any desired procedure.

In a preferred embodiment, the nucleic acid comprises an expressiblegene which is functional in the target cell. For example, the gene mayencode coagulation factors, (such as Factor IX), enzymes involved inspecific metabolic defects, (such as urea cycle enzymes, especiallyornithine transcarbamylase, argininosuccinate synthase, and carbamylphosphate synthase); receptors, (e.g., LDL receptor); toxins; thymidinekinase to ablate specific cells or tissues; ion channels (e.g., chloridechannel of cystic fibrosis); membrane transporters (e.g., glucosetransporter); and cytoskeletal proteins, (e.g., dystrophin). The genemay be of synthetic, cDNA or genomic origin, or a combination thereof.The gene may be one which occurs in nature, a non-naturally occurringgene which nonetheless encodes a naturally occurring polypeptide, or agene which encodes a recognizable mutant of such a polypeptide. It mayalso encode an mRNA which will be "antisense" to a DNA found or an mRNAnormally transcribed in the host cell, but which antisense RNA is notitself translatable into a functional protein.

For the gene to be expressible, the coding sequence must be operablylinked to a promoter sequence functional in the target cell. Two DNAsequences (such as a promoter region sequence and a coding sequence) aresaid to be operably linked if the nature of the linkage between the twoDNA sequences does not (1) result in the introduction of a frame-shiftmutation in the region sequence to direct the transcription of thedesired gene sequence, or (3) interfere with the ability of the genesequence to be transcribed by the promoter region sequence. A promoterregion would be operably linked to a DNA sequence if the promoter werecapable of effecting transcription of that DNA sequence. In order to be"operably linked" it is not necessary that two sequences be immediatelyadjacent to one another. A nucleic acid molecule, such as DNA, is saidto be "capable of expressing" a mRNA if it contains nucleotide sequenceswhich contain transcriptional regulatory information and such sequencesare "operably linked" to nucleotide sequences which encode the RNA. Theprecise nature of the regulatory regions needed for gene expression mayvary from organism to organism, but in general include a promoter whichdirects the initiation of RNA transcription. Such regions may includethose 5'-non-coding sequences involved with initiation of transcriptionsuch as the TATA box.

If desired, the non-coding region 3' to the gene sequence coding for thedesired RNA product may be obtained. This region may be retained for itstranscriptional termination regulatory sequences, such as those whichprovide for termination and polyadenylation. Thus, by retaining the3'-region naturally contiguous to the coding sequence, thetranscriptional termination signals may be provided. Where thetranscriptional termination signals are not satisfactorily functional inthe expression host cell, then a 3' region functional in the host cellmay be substituted.

The promoter may be an "ubiquitous" promoter active in essentially allcells of the host organism, e.g., for mammals, the beta-actin promoter,or it may be a promoter whose expression is more or less specific to thetarget cells. Generally speaking, the latter is preferred. A promoternative to a gene which is naturally expressed in the target cell may beused for this purpose, e.g. the PEPCK (phosphoenol pyruvatecarboxykinase) promoter for expression in mammalian liver cells. Othersuitable promoters include albumin, metallothionein, surfactant, apoE,pyruvate kinase, LDL receptor HMG CoA reductase or any promoter whichhas been isolated, cloned and shown to have an appropriate pattern oftissue specific expression and regulation by factors (hormones, diet,heavy metals, etc.) required to control the transcription of the gene inthe target tissue. In addition, a broad variety of viral promoters canbe used; these include MMTV, SV-40 and CMV. An "expression vector" is avector which (due to the presence of appropriate transcriptional and/ortranslational control sequences) is capable of expressing a DNA (orcDNA) molecule which has been cloned into the vector and of therebyproducing an RNA or protein product. Expression of the cloned sequencesoccurs when the expression vector is introduced into an appropriate hostcell. If a prokaryotic expression vector is employed, then theappropriate host cell would be any prokaryotic cell capable ofexpressing the cloned sequences. Similarly, when a eukaryotic expressionvector is employed, then the appropriate host cell would be anyeukaryotic cell capable of expressing the cloned sequences.

In addition to or instead of an expressible gene, the nucleic acid maycomprise sequences homologous to genetic material of the target cell,whereby it may insert itself ("integrate") into the genome by homologousrecombination, thereby displacing a coding or control sequence of agene, or deleting a gene altogether.

In another embodiment, the nucleic acid molecule is "antisense" to agenomic or other DNA sequence of the target organism (including virusesand other pathogens) or to a messenger RNA transcribed in cells of theorganisms, which hybridizes sufficiently thereto to inhibit thetranscription of the target genomic DNA or the translation of the targetmessenger RNA. The efficiency of such hybridization is a function of thelength and structure of the hybridizing sequences. The longer thesequence and the closer the complementarily to perfection, the strongerthe interaction. As the number of base pair mismatches increases, thehybridization efficiency will fall off. Furthermore, the GC content ofthe packaging sequence DNA or the antisense RNA will also affect thehybridization efficiency due to the additional hydrogen bond present ina GC base pair compared to an AT (or AU) base pair. Thus, a targetsequence richer in GC content is preferable as a target.

It is desirable to avoid antisense sequences which would form secondarystructure due to intramolecular hybridization, since this would renderthe antisense nucleic acid less active or inactive for its intendedpurpose. One of ordinary skill in the art will readily appreciatewhether a sequence has a tendency to form a secondary structure.Secondary structures may be avoided by selecting a different targetsequence.

An oligonucleotide, between about 15 and about 100 bases in length andcomplementary to'the target sequence may be synthesized from naturalmononucleosides or, alternatively, from mononucleosides havingsubstitutions at the non-bridging phosphorous bound oxygens. A preferredanalogue is a methylphosphonate analogue of the naturally occurringmononucleosides. More generally, the mononucleoside analogue is anyanalogue whose use results in oligonucleotides which have the advantagesof (a) an improved ability to diffuse through cell membranes and/or (b)resistance to nuclease digestion within the body of a subject (Miller,P. S. et al., Biochemistry 20:1874-1880 (1981)). Such nucleosideanalogues are well-known in the art. The nucleic acid molecule may be ananalogue of DNA or RNA. The present invention is not limited to use ofany particular DNA or RNA analogue, provided it is capable of fulfillingits therapeutic purpose, has adequate resistance to nucleases, andadequate bioavailability and cell take-up. DNA or RNA may be made moreresistant to in vivo degradation by enzymes, e.g., nucleases, bymodifying internucleoside linkages (e.g., methylphosphonates orphosphorothioates) or by incorporating modified nucleosides (e.g.,2'-0-methylribose or 1'-alpha-anomers).

The naturally occurring linkage is ##STR1##

Alternative linkages include the following: ##STR2##

It is also possible to replace the 3'O-P-O5' with other linkages such as3'O-CH₂ C(O)-O5', 3'O-C(O)-NH5', and 3'C- CH₂ CH₂ S-C5'.

The entire nucleic acid molecule may be formed of such modifiedlinkages, or only certain portions, such as the 5' and 3' ends, may beso affected, thereby providing resistance to exonucleases.

Nucleic acid molecules suitable for use in the present invention thusinclude but are not limited to dideoxyribonucleoside methylphosphonates,see Mill, et al., Biochemistry, 18:5134-43 (1979), oligodeoxynucleotidephosphorothioates, see Matsukura, et al., Proc. Nat. Acad. Sci.,84:7706-10 (1987), oligodeoxynucleotides covalently linked to anintercalating agent, see Zerial, et al., Nucleic Acids Res., 15:9909-19(1987), oligodeoxynucleotide conjugated with poly(L-lysine), seeLeonetti, et al., Gene, 72:32-33 (1988), and carbamate--linked oligomersassembled from ribose-derived subunits, see Summerton, J., AntisenseNucleic Acids Conference, 37:44 (New York 1989).

Compaction of the Nucleic Acid

It is desirable that the complex of the nucleic acid and the nucleicacid binding moiety be compacted to a particle size which issufficiently small to achieve uptake by receptor mediated endocytosis,passive internalization, receptor-mediated membrane permeabilization, orother applicable mechanisms. Desirably, the complex of the compactednucleic acid, the target binding moiety, and the nucleic acid bindingmoiety is small, e.g., less than 100 nm, because the sinusoidalcapillary systems of the lung and spleen will trap aggregates of thatsize, and more preferably less than 80 or 90 nm, as that is the typicalinternal diameter of coated-pit endocytic vesicles.

Since complexes larger than 30 nm may be susceptible to nonspecifictakeup by macrophages in the spleen and liver, the conjugate ispreferably also smaller than 30 nm.

In the case of the ASGP receptor of the liver, complexes larger than15-23 nm are excluded from uptake. This size limitation in vivo for thereceptor is probably directly related to the existence of anotherreceptor for galactosylated proteins in the Kupffer cells of the liver.The Kupffer cell receptor is very efficient in taking up and degradinggalactosylated molecules of larger size in vivo and thus, would competefor the uptake of the galactosylated DNA complex with the ASGP receptoron the surface of hepatocytes. Most preferably, for liver delivery, thecomplex is less than 23 nm, more preferably less than 15 nm, still morepreferably no more than 12 nm in diameter.

The present invention calls for the complex of the nucleic acid and thenucleic acid-binding carrier to be compacted without causing aggregationor precipitation, and preferably to a condensed state (see FIG. 12). Forthe purpose of the present invention, it is helpful to characterize DNAas having one of the following states: normal (uncondensed); condensed;relaxed; uni-aggregated (clusters of unimolecular toroids);multi-aggregated (clusters of multimolecular toroids); and precipitated.These states are defined in terms of their appearance under electronmicroscopy (see Table 103).

Condensed DNA is in a state in which interaction with the solvent isminimal and therefore the DNA is in the form of isolated spheres ortoroids. It is not fibrous to an appreciable degree. Relaxed DNA,typically formed by dissociation of polycation from the DNA, formsfibers. Aggregated DNA forms clumped or multimolecular toroids.

The theoretical size of a unimolecular DNA complex can be calculated bythe formulae set forth in legends "b" and "c" of Table 106. Preferably,the complexes of this invention have a diameter which is less thandouble the size calculated by one or both of these formulae. Largercomplexes are likely to correspond to multimolecularly aggregated DNA.

DNA can be compacted to a condensed state by neutralizing its charge,e.g., by addition of a polycation, or otherwise reducing itsinteractions with solvent. However, the polycation can cause aggregationor precipitation of the DNA if a chaotropic agent is not employed toprevent it. Compaction therefore can be accomplished by judicious use ofboth the polycation (to condense the DNA) and (as needed) of achaotropic agent (to prevent aggregation or precipitation).

Overuse of the chaotropic agent can, however, result in relaxation ofthe DNA. Preferably, the complex has a unaggregated, unimolecular toroidstructure condensed to smaller than 23 nm in diameter; the degree ofcompaction may be determined by electron microscopy. For example, acomplex of the PEPCK-hFIX gene with galactosylated polylysine has beencompacted to a unimolecular toroid with a mean diameter of about 12 nm,as shown in Table 106.

The term "unimolecular toroid" indicates that the toroid contains onlyone nucleic acid molecule; the toroid may contain many carrier (e.g.,galactosylated poly-Lys) molecules. A typical ratio is one DNA moleculeto about 100 carrier molecules, per "unimolecular" toroid.Alternatively, and perhaps more precisely, this structure may bereferred to as a mono-nucleic acid toroid. Unimolecular andmultimolecular toroids (the latter each contain more than one DNAmolecule) may be distinguished by the different size of each of thecomplexes when viewed by the electron microscope, indicating the multi-or unimolecular (counting only the DNA molecules) composition of thetoroids.

We have also used other techniques to identify structural changes in theDNA upon poly-L-lysine binding. The first of these is thespectrophotometric determination of the turbidity in the solution usingthe absorbance at 400 nm. Turbidity is primarily an indicator ofaggregation. Aggregation is confirmed by a circular dichroism (CD) valuegreater than 0 at wavelengths from 300 to 340 nm.

FIG. 18 illustrates the effect on turbidity of adding the poly-L-lysineto the DNA solution at different starting concentrations of NaCl.Turbidity increases as the initial concentration of salt is increased(this could be easily confirmed by eye), indicating that thecondensation of the DNA complex at lower ionic strength results in asuspension of particles composed of unimolecular DNA-poly-L-lysinecomplexes interacting with each other. We noted that the solutions ofDNA condensed at lower salt concentration were clear, with the presenceof particulate matter in suspension. Solutions containing the DNAcomplex with different degrees of turbidity were analyzed by EM tovisualize the DNA structures formed in each situation. Appropriatelycondensed, unimolecular DNA complexes were found with both clear andslightly turbid solutions. This was not true for the condensation of DNAcomplexes at initial low ionic strength where we noted minimalabsorbance at 400 nm (FIG. 18) because the solutions containingparticles in suspension did not absorb at 400 nm. However, when thesesolutions were analyzed using EM, we noted the expected transitionalstructures shown in FIG. 1. When the particles in suspension becametotally dispersed, the structures identified by EM were essentiallyidentical to condensed unimolecular DNA complexes. Thus, turbidity ofthe solution containing the DNA complexes is dependent on the initialconcentration of salt used for condensation of the complex. Although themechanisms responsible for the observed differences in the condensationof DNA at initial low and high ionic strength is not clear, we adaptedour protocol to appropriately condense DNA, avoiding the formation ofturbid solutions.

A more reliable technique for diagnosing the structural transition ofDNA-poly-L-lysine complexes in solution is the absorbance of thecondensing complex at 260 nm as the concentration of NaCl increases. Theuni-aggregated DNA complex in suspension has only 10-30% of the expectedabsorbance because the particulate matter does not absorb at 260 nm. Theaddition of NaCl disperses the uni-aggregated DNA complex in suspensionwhich results in the observed steep increase in the absorbance noted inFIG. 11. At this point the solution is clear and there are no visibleparticulate structures in suspension. This feature of theDNA-poly-L-lysine condensation clearly correlates with the structuresshown in FIG. 1. At a concentration of NaCl which causes a steepincrease in the absorbance at 260 nm, we observed unaggregated,condensed complexes by EM; before this critical concentration of NaClwas attained, the DNA complex appear aggregated and at higher NaClconcentrations the DNA complex was relaxed. A second transition inabsorbance at 260 nm, as a result of the relaxation of the condensed DNAcomplex that was in suspension, indicates the full solubilization of theDNA complex.

Circular dichroism (CD) can be used to monitor the condensation of DNA.When the spectrum is identical to that of DNA alone, then the DNAcomplex is assumed to be correctly compacted, i.e., into unimolecularcomplexes. In another words, the positive spectrum at 220 nm isquantitatively similar to the 220 nm spectrum of DNA alone, and thecross-over (the wavelength at which the spectrum of the complex crossesthe 0 point) is essentially identical to that of DNA alone. When the DNAaggregates into multimolecular complexes, the positive spectrum at 270nm is inverted into a negative spectrum at that wavelength (this iscalled psi-DNA structure or ψ-DNA).

Table 103 sets forth the characteristics of each state as determined bynaked eye observation, circular dichroism spectroscopy, electronmicroscopy, and absorbance at 260 nm. I t should be noted that any othertechniques which are capable of identifying condensed DNA complexes maybe used instead of or in combination with those discussed above.

To compact the nucleic acid, the carrier is added to the nucleic acidsolution, whereby the carrier disrupts the nucleic acid: solventinteractions allowing the nucleic acid to condense. Preferably, at leastthe turbidity of the solution is monitored as the carrier is added, sothat a change in state is promptly detected. Once turbidity appears, thestate of the DNA may be further analyzed by CD spectroscopy to determinewhether the DNA is in the condensed or the aggregated state.(Precipitation should also be detectable with the naked eye.)Preferably, the carrier is added sufficiently slowly to the nucleic acidsolution so that precipitation and aggregation are minimized. Ifprecipitation or aggregation occur, a chaotropic salt should be addedslowly, and the result again examined by CD spectroscopy. The preferredsalt is NaCl. Other chaotropic salts can be used as long as they aretolerated by the animal (or cells) to which they will be administered.Suitable agents include Sodium sulfate (Na₂ SO₄). Lithium sulfate (Li₂SO₄), Ammonium sulfate ((NH₄)₂ SO₄, Potassium sulfate (K₂ SO₄),Magnesium sulfate (MgSO₄), Potassium phosphate (KH₂ PO₄), Sodiumphosphate (NaH₂ PO₄), Ammonium phosphate (NH₄ H₂ PO₄), Magnesiumphosphate (MgHPO₄), Magnesium chloride (Mg Cl₂), Lithium chloride(LiCl), Sodium chloride (NaCl), Potassium chloride (KCl), Cesiumchloride (CaCl), Ammonium acetate, Potassium acetate, Sodium acetate,Sodium fluoride (NaF), Potassium fluoride (KF), Tetramethyl ammoniumchloride (TMA-Cl), Tetrabutylammonium chloride (TBA-Cl),Triethylammoniym chloride (TEA-Cl), and Methyltriethylammonium chloride(MTEA-Cl)

We have investigated variables that affect condensation of DNA in vitroand the functional relevance of these parameters for efficient deliveryof DNA complexes into animals by receptor-mediated endocytosis. We noteda strong correlation between the ionic strength at which the condensedDNA-poly-L-lysine complex remains stable in solution and theconcentration of DNA. These experiments were performed using a 4.5 kbplasmid containing the promoter from the gene for PEPCK linked to thestructural gene for hFIX, using a ratio of DNA to poly-L-lysine thatresulted in a 1 to 1 ratio of negative to positive charges in solution.The variation in the final concentration of NaCl necessary to solubilizethe particles is a logarithmic function of DNA concentration, in whichthe condensation of highly concentrated DNA-poly-L-lysine complexesoccurs with only a slight increase in ionic strength. This physicalcharacteristic of DNA condensation has clear advantages for the deliveryof the DNA particles to tissues of adult animals in vivo since it haslittle effect on the ionic load in the animal's blood.

The linear fit of the data using the least square method is described bythe following function: ##STR3## where b0=2.52×10E-3, b1=0.577

We have observed variations in the function described by the aboveequation when we use different DNA plasmids and different DNApreparations during the condensation process. These differences areprobably related to the variation in the affinity of poly-L-lysine forDNA of different sources and compositions. For maximum binding affinitywe generally use DNA precipitated twice with sodium acetate and 2.5volumes of -40 C. ethanol (see Methods). We have not found an apparentdifference in binding affinity of poly-L-lysine for DNA of differentforms (i.e. supercoiled, nicked and linear) and for DNA extracted usinganionic exchange chromatography or cesium chloride gradientcentrifugation. This may indicate the presence of a contaminant in theDNA preparations from different sources which has poly-L-lysine bindingactivity, that is eliminated by sequential DNA precipitation.

We have also investigated the effect of the length of the poly-L-lysineon the concentration of NaCl necessary for the effective condensation ofDNA (FIG. 19). The correlation between these variables was assessedusing a fixed concentration of DNA from different sources. We have useda broad range of poly-L-lysine lengths; essentially the sizes ofpoly-L-lysine available commercially. However, the length of thepoly-L-lysine in an average of various sizes of the protein asdetermined by low-angle light scattering analysis of individual lots ofchemically synthesized poly-L-lysine. The actual distribution of sizeswithin each sample varies from 60 to 80% of the material beingdistributed, which is +/- 20% from the average size. This broaddistribution within a single size is a source of error in ourdeterminations. Nevertheless, there is a clear correlation observable inFIG. 19 between the length of the poly-L-lysine and the necessaryconcentration of NaCl needed for the condensation of the DNA complex insolution. This correlation is a linear function of poly-L-lysine lengthup to a size of 150 lysine residues, after which the function reachessaturation and there is no increase in the concentration of NaCl neededfor the condensation of DNA with longer poly-L-lysine. These data areconsistent with a cooperative binding between the poly-L-lysine and theDNA phosphate backbone. Thus, by reducing the length of thepoly-L-lysine molecules used to condensed the DNA the solution of DNAcomplex injected into the animals will be less hypertonic. It is alsoimportant to consider the dilution of the DNA complex in the blood ofthe animal to evaluate the functional significance of these changes inionic strength on the efficiency of this method for gene therapy. Wehave injected rats with DNA complexes containing longer range ofpoly-L-lysine lengths than those shown in FIG. 19 and rabbits with theshorter range of sizes of poly-L-lysine, and noted positive andpersistent expression of the transfected genes in both cases.

The preferred minimum initial salt concentration is dependent on thecompaction activity of the carrier and the chaotropic activity of thesalt. If the NABM were (Lys)₈, or (Lys)₂₇, the initial NaClconcentration could be zero. With longer polyols chains, however, in theabsence of NaCl, precipitation would be immediate. With (Lys)₅₀, theinitial NaCl concentration is preferably be at least about 300 mM.Nonetheless, if the TBM is a protein that affects the condensation, theinitial salt concentration could be as low as zero.

The carrier may be added continuously, or in small discrete steps. Onemay begin with a higher flow rate, or larger aliquots, and reduce theflow rate or aliquot size as the desired endpoint of the reaction isneared. Typically 0.1 to 10% of the carrier solution is added at a timeto the DNA solution. Each addition is preferably made every 2 seconds to2 minutes, with constant vortexing. However, longer settlement times maybe allowed.

In one embodiment, a nucleic acid, contained in a salt solution, whichis preferably at least 0.5M, but less than 1.5M NaCl, is mixed withpoly-L-lysine (109 lysines) containing the covalently linked target cellbinding moiety (for example, galactose), which is contained in asolution of NaCl at the same concentration (e.g., 0.5 to 1.5M NaCl).Preferably, the molar ratio of nucleic acid phosphate group topositively charged group of the DNA binding moiety is in the range of4:1 to 1:4, and more preferably is about 1.5:1.

Some of Applicants' experimental results are set forth in Table 104. Wehave taken 16 examples (asterisked in the first column of Table 104)which were tested and worked in vivo, and regressed final NaClconcentration (the independent variable) against DNA concentration andpoly-L-Lys length (the dependent variables), with the results given inTable 105.

The Conjugation

In the embodiments relying on a target-binding carrier molecule, thenucleic acid binding moiety will be conjugated, covalently ornoncovalently, directly or indirectly, to the target cell bindingmoiety. The conjugation may be performed after, or, more usually before,the loading of the nucleic acid binding moiety with the nucleic acid ofinterest. Either way, the conjugation should not substantially interferewith the binding of the nucleic acid to the nucleic acid binding moiety,or, for that matter, with the ability of the target cell binding moietyto bind to the target cell.

Pharmaceutical Compositions and Methods

The compacted nucleic acid, optionally conjugated with a TBM, may beadmixed with a pharmaceutically acceptable carrier for administration toa human or other animal subject. It will be appreciated that it ispossible for a DNA solution to contain both condensed DNA and relaxedDNA. The compositions of this invention preferably are sufficiently richin condensed complexes so that the absorbance at 260 nm is less than 50%that of naked DNA of equal concentration. As stated in Table 103,condensed DNA usually has an absorbance of 20-30%, and relaxed DNA,80-100%, that of naked DNA.

The administration may be by any suitable route of administration. Thedosage form must be appropriate for that route. Suitable routes ofadministration and dosage forms include intravascular (injectablesolution), subcutaneous (injectable solution, slow-release implant),topical (ointment, salve, cream), and oral (solution, tablet, capsule).With some routes of administration, the dosage form must be formulatedto protect the conjugate from degradation, e.g., by inclusion of aprotective coating or of a nuclease inhibitor.

The dosage may be determined by systematic testing of alternative doses,as is conventional in the art.

Rats (200-300 g) tolerate as much as 600 μg doses of the DNA complex ofExample 1 without any apparent ill effects on growth or health. Mice (25g) have been injected with 150 μg of that DNA complex without anyapparent problem.

In humans, a typical trial dose would be 60-120 mg of DNA; if this doseis too low to be effective or so high as to be toxic, it may beincreased, or decreased, respectively, in a systematic manner, until asuitable dose is identified.

For short life span cells, e.g., macrophages, a typical dosing schedulemight be one dose every two weeks. For long life span cells, e.g.,hepatocytes, one dose every two months might be preferable.

Adjuvants may be used to decrease the size of the DNA complex (e.g. 2-10mM MgCl), to increase its stability (e.g., sucrose, dextrose, glycerol),or to improve delivery efficiency (e.g., lysosomotropic agents such aschloroquine and monensine). The complexes may be enclosed in a liposometo protect them and to facilitate their entry into the target cell (byfusion of the liposome with the cell membrane).

The invention is illustrated, but not limited, by the followingexamples.

EXAMPLE 1

Introduction

Christmas disease, or Hemophilia B, is a sex-linked recessive bleedingdisorder due to a deficiency of functional coagulation factor IX in thecirculation. Human factor IX (hFIX) is a plasma glycoprotein normallysynthesized in the liver, that plays an integral role in the intrinsiccoagulation pathway. Once it has been converted to its serine proteaseform (IXa) by activated plasma thromboplastin antecedent (factor XIa),the activated protein interacts with coagulation factor VIIIa, calciumions, and phospholipids to produce a complex that converts factor X toXa. Factor IX undergoes several post-translational modifications in theliver that are essential for its function before secretion into theblood. These include Vitamin K dependent γ-carboxylation ofamino-terminal glutamic acid residues and β-hydroxylation of asparticacid.

Christmas disease accounts for approximately 10 to 20 percent of allinherited clotting disorders. Affected individuals exhibit a wide rangeof clinical severity that generally correlates with the level ofcirculating factor IX. Patients with severe deficiencies of functionalfactor IX may bleed spontaneously into soft tissues and joints or afterminor trauma. Transfusions of plasma or concentrates rich in factor IXare used to abort bleeding episodes by temporarily correcting thedeficiency. Unfortunately, clinical management has been confounded byviral contamination of pooled plasma. Blood-borne infections, such ashepatitis and the acquired immunodeficiency syndrome, have becomesignificant problems in the treatment of the hereditary clottingdisorders. These complications stress the importance of developingalternative treatments.

The gene for human coagulation factor IX has been identified andsequenced; 1,248 base pairs, in length, the complementary DNA predicts aprotein of 416 amino acids, and, after post-translational modifications,the mature protein has a molecular weight of approximately 54,000 Da. Agene encoding human coagulation factor IX may be used for geneticcorrection of hemophilia B.

A chimeric P-enolpyruvate carboxykinase-human factor IX(PEPCK-hFIX) gene(50% supercoiled/ 50% open circular) was condensed with galactosylatedpoly-L-lysine (average length 50 or 109 amino acids) by titration withNaCl. This process was monitored using CD spectroscopy and electronmicroscopy and resulted in the formation of a DNA-carrier complex of10-12 nm in diameter at a critical NaCl concentration. We haveintroduced the PEPCK-hFIX gene, conjugated using this procedure, intothe intact livers of adult rats and have demonstrated that theDNA-carrier complex specifically targets the gene to this organ and thathFIX DNA, MRNA and hFIX protein can be demonstrated up to 140 days (theduration of the experiment) after administration of the DNA-carriercomplex. The gene is present as an episome as determined by Southernanalysis of DNA isolated from the liver of an animal 32 days afterinjection of the DNA-conjugate. Transcription of the PEPCK-hFIX gene wascontrolled by diet for the entire time course of the experiment; feedingthe animals a carbohydrate-free diet for one week resulted in thepredicted induction of hFIX in the blood, as detected by Western blothybridization.

Methods

A. Galactosylation

Polymers of L-lysine-HBr or L-lysine-Cl with an average chain length of109 (Sigma) were galactosylated essentially as described by Monsigny, etal. (1984) Biol. Cell., 51, 187. Briefly, 2 mg of poly-L-lysine wasreacted with 89 g of α-D-galactopyranosyl phenyl-isothiocyanate (SigmaG-3266) dissolved in N,N-Dimethyl formamide (5 mg/ml). The solution wasadjusted to pH 9.0 by the addition of 1/10 volume of 1M sodium carbonatepH 9.0. Since the reaction is 10% efficient, 0.8% of the ε-NH₃ groupspresent in the solution are glycosylated. The tube was shielded fromlight by aluminum foil and mixed for 6 hours at room temperature. Thesolution was then dialyzed, using Spectra-Por dialysis tubing (Fisher3500 M.W. cutoff), against 500 ml of 5 mM NaCl buffer for 2 days withfrequent changes of buffer (2 changes/day).

B. Analysis of the ligand

The dialyzed solution was then analyzed spectrophotometrically at 205 Åand 250 Å for the concentration of poly-L-lysine and the concentrationof phenyl-galactose residues, respectively. This step ensures thatsignificant losses during dialysis have not occurred, and that thegalactosylation reaction was complete, since in the solution only themodified galactose will absorb at 250 Å.

C. Complex formation

Plasmid DNA was prepared using standard techniques. The DNA wasre-suspended in 10 mM Tris-HCl, pH 8.0, containing 1 mM EDTA and theconcentration of the DNA determined spectrophotometrically. The DNApreparation was digested twice with RNAses A+T1. This step ensures thatRNA is not present in the solution (RNA inhibits the condensation of DNAby poly-L-lysine). A solution containing a high concentration of DNA(1.5-2 mg/ml) was used in further steps. An example of a typicalprotocol for DNA condensation is described as follows:

a) 300 μg of DNA in 200 μl of 0.75M NaCl (added from 5M NaCl solution)is vortexed at medium speed, using a VIBRAX machine (IKA-VIBRAX-VXR).This procedure is desirable to increase the effective length of the DNApolymer in high salt solutions, thus achieving efficient binding of thepoly-L-lysine moiety to the DNA backbone.

b) 84 μg of poly-L-lysine-galactose in 200 μl of 0.75M NaCl (added froma 5M NaCl solution) is added dropwise over a period of 30 minutes to 1hour in 20 μl aliquots. This amount translates into a molar ratio of 1DNA PO₄ ⁻ group to 0.7 carrier NH₃ ⁺ groups.

c) The solution becomes turbid at the end of the process. 3 μl aliquotsof 5M NaCl are added dropwise to the vortexing solution until turbiditydisappears as monitored by eye. This process is slow, allowing 30seconds between the addition of each new aliquot of 5M NaCl. Then thesolution is subjected to CD spectroscopic monitoring while 2 μl aliquotsof 5M NaCl are gradually added. The condensation process is completewhen the diagnostic spectrum of the DNA complex is observed. Forsubsequent preparations of DNA complex consisting in the same plasmidDNA at the same concentration of nucleotide, the protocol can befollowed without monitoring with CD and the results will be fullyreproducible. When using different concentration of DNA or a differentplasmid the CD monitoring should be repeated.

We have found that an alternative technique for monitoring DNA complexformation gives similar results. This technique consists of thefollowing steps:

a) and b) Idem.

c) The solution becomes turbid at the end of the process. 3 μl aliquotsof 5M NaCl are added dropwise to the vortexing solution until turbiditydisappears as monitored by eye. This process is slow, allowing 30seconds between the addition of each new aliquot of 5M NaCl. Thesolution is then centrifuged at full speed (12000× g) for 30 secondsusing a microcentrifuge and the appearance of precipitate is monitored.If a precipitate is observed 2 μl aliquots of 5M NaCl are added. Thesolution is further vortexed for 0.5 minutes and the centrifugation stepis repeated. The appearance of a precipitate is due to the aggregationof the DNA-complex in solution and indicates that the DNA has not beenfully collapsed.

Results and Discussion

In developing the procedure described herein, we have monitored thephysical structure of the DNA/ligand-poly-L-lysine conjugate usingcircular dichroism (CD) and electron microscopy and studied theconditions by which a functional complex is generated. We thendetermined the functional relevance of the physical structure of theDNA/ligand-poly-L-lysine conjugate using intact animals. The DNA wascondensed by the addition of the ligand-poly-L-lysine in the presence ofvarying concentrations of NaCl. Either 60 μg of RNA-free CMV-β-galactosidase (A) or phFIX (B,C,D, and E), diluted to a final volumeof 150 μl in 700 mM NaCl were vortexed at medium speed in a VIBRAXapparatus (IKA-VIBRAX-VXR). 19 μg of α-galactopyranosyl-phenylisothiocyanate/poly-L-lysine biconjugate (Sigma) were diluted in thesame way and added dropwise to the vortexing solution of DNA. For invivo studies, 300 μg of DNA (dissolved in TE buffer, pH 8) in 150 μl of700 mM NaCl were condensed with 95 μg of α-galactopyranosyl-phenylisothiocyanate/poly-L-lysine biconjugate in 150 μl of 700 mM NaCl. Theslow addition of the polycation results in the formation of a turbidsolution which is dissolved by the stepwise addition of 3 μl aliquots of5M NaCl. The disappearance of the turbidity was monitored by eye and atthe point of no turbidity the solutions of DNA/poly-L-lysine complexeswere investigated by both electron microscopy (E.M.) and CDspectroscopy. Continuing addition of. 2 μl aliquots of 5M NaCl resultedin structural changes as shown in FIGS. 1A-1F. Representative spectrademonstrating different structural conformations of the DNA complex atvarious concentrations of NaCl and in the presence and absence of addedpoly-L-lysine, are presented in FIG. 1. Polycation binding to DNAresults in a specific spectrum characterized by a displacement of thecross-over to longer wavelengths; this shift can be correlated with thechiral packing of DNA/poly-L-lysine conjugates in high order, asymmetricstructures similar to the Y-form of DNA. As shown in FIG. 1A, doublestranded DNA (in 1M NaCl) has a characteristic spectrum which wasmarkedly altered by the addition of poly-L-lysine at varying ionicstrengths. (FIG. 1a). When the ionic strength of theDNA/ligand-poly-L-lysine conjugate was increased the complex proceededthrough a transition from an aggregated (FIG. 1C) to a condensed state(FIG. 1D & FIG. 1E). This corresponds to a shift in the spectrum of thecomplex as shown in FIG. 1A. The change in the CD spectra at 220 nm andthe shift in the cross-over (0 line in FIG. 1A) that occurs withincreasing ionic strength of the solution is of particular importance inmonitoring the formation of condensed DNA complex by means of CDspectroscopy. If the ionic strength is increased above the criticalrange required for the condensation of the DNA complex, the complexassumes a non-condensed, relaxed conformation (FIG. 1F). This transitionin the conformation of the DNA complex cannot be monitored by CDspectroscopy so that a rigorous titration of NaCl is critical to thesuccess of this procedure. It is important to note that the diameter ofthe DNA complex observed in FIG. 1D (about 10 nm) conforms with thediscrimination range desirable for internalization of molecular ligandsby the hepatic receptor for asialoglycoproteins.

We therefore verified the functional relevance of the observed DNAstructures as vehicles to transfer of the DNA into hepatocytes in vivoby receptor-mediated endocytosis. In order to establish the nature ofthe uptake process, we followed the removal of the DNA complex from themedia by HepG2 cells, which contain the asialoglycoprotein receptor. Theuptake of the DNA complex was completely inhibited when a 100-fold molarexcess asialogetuin was used as a competitor, indicating that thecomplex was being taken up by receptor-mediated endocytosis via theASGP.

A plasmid (pPFIX) containing a chimeric gene composed of the promoter ofthe gene for the cytosolic form of P-enolpyruvate carboxydinase (PEPCK)from the rat, linked to the cDNA for human coagulation Factor IX (hFIX)(Ferkol, et al., FASEB J., 7:1081 (1993)) was used to follow thedelivery and expression of the DNA in the liver. The time-course ofexpression of hFIX gene in the transfected animals was determined byWestern blot hybridization, using a monoclonal antibody against themature hFIX peptide.

Adult, male Sprague-Dawley rats, approximately 250 g in weight, wereanesthetized with ether. 300-400 μl of a solution containing 300 μg ofPPFIX complexed as previously described with galactose-poly-L-lysine,were infused into the caudal cava vein. Rats were killed at 0, 4, 8, 12,32, 72 and 136 days after transfection and tissues and blood samplestaken.

Plasma samples (1 μl) from transfected animals and a 1:4 dilution of ahuman plasma control were subjected to electrophoresis in SDS/10%polyacrylamide gels and transferred onto nitrocellulose membrane filtersusing standard techniques. the blots were block with 1× PBS, pH 7.4,0.03% polyoxyethylene sorbitan monolaurate (Tween 20), and 10% (w/v) dryskim milk for two hours at room temperature, followed by incubation witha 1/1000 dilution of a monoclonal murine anti-human factor IX antibody(3 μg/ml) for two hours at room temperature. The monoclonal antibody waskindly provided by Dr. Kenneth Smith (United Blood Services,Albuquerque, N.Mex.). The membrane was washed three times in 1× PBS, pH7.4 and 0.039% Tween 20, then incubated with a 1/500 dilution of goatanti-murine lgg (H+L)--horseradish peroxidase conjugate. The membranewas then washed vigorously four times with 1× PBS, pH 7.4 and 0.03%Tween 20, and 10 ml of Western blot enhanced chemiluminescence detectionsolution was applied for one minute. The luminescence emitted from thefilter was detected by a 20 second exposure to photographic film. Wedetected a band hybridizing specifically to the hFIX monoclonal antibodyfor as long as 140 days. No hybridizing band was detected inuntransfected controls.

The liver from an animal 32 days after transfection was taken andgenomic DNA isolated using standard techniques. 5 μg of total DNA fromthe transfected animal and from a nontransfected control were digestedwith either EcoRI or BgI II overnight. Southern blot electrophoresis wasperformed by established methods. The DNA from the transfected animalonly hybridized to 4.5 kb BglII and a 2.6 kb EcoRI probes.

Spleen, lung, heart and liver tissues were obtained from a rattransfected with 300 μg of the DNA complex. PCR analysis was carried outon total genomic DNA isolated from these tissues. Only the liver of thetransfected rat, and not its spleen, lung or heart, or the liver of acontrol animal, was positive for the 720 bp probe.

The presence of mRNA transcripts for human factor IX in the livers ofrats transfected with pFIX was determined after treatment of totalcellular hepatic RNA with Moloney Murine Leukemia virus reversetranscriptase and amplification of the resultant cDNA by the polymerasechain reaction. Briefly, 1 μg of total rat liver RNA was treated with 10U DNAse I (RNAse free), and added to a solution containing 500 nM of(dT)₁₆ oligonucleotide primer and 500 nM of each dNTP, and heated to 42°C., and 1 μl of the cDNA pool was amplified by the polymerase chainreaction, using primers expanding the 5' UTR region of the PEPCKpromoter and the cDNA for hFIX. As a control, the same RNA samples notconverted to cDNA by reverse transcriptase were also used as polymerasechain reaction templates to ensure that contaminating plasmid DNA hadnot been amplified. The products were separated by agarose gelelectrophoresis and Southern blot hybridization using a radiolabeledhuman factor IX cDNA probe. We observed a band that hybridizedspecifically with the hFIX probe only in the transfected animals. Nobands were detected in either non-transfected controls or transfectedsamples not converted to cDNA by reverse transcriptase.

The functional activity of hFIX in the plasma of transfected animals wasanalyzed by measuring the procoagulant activity of human Factor IX. Amodification of the one stage, kaolin-activated, partial thromboplastintime with factor IX-deficient human plasma was used. Blood samples wereobtained from experimental animals by venipuncture. One fiftieth volumeof 500 mM sodium citrate pH 5.0, was added to prevent coagulation, andthe plasma was stored at 20° C. The samples were assayed in duplicate,and their activity ws compared to the functional activity of pooledplasma from 24 normal adult human males. In normal human plasma isequivalent 100% functional activity or approximately 3 μg of humanFactor IX per ml. Background Factor IX activity in rat plasm(approximately 0.15 units/ml of Factor IX activity in rat serum) wassubtracted from each value of human Factor IX determined in individualanimals. The background values is non-specific cross activity of ratFactor IX determined in the human Factor IX assay used in this analysis.Blood samples were obtained from experimental animals by venipuncture.One fiftieth volume of 500 mM sodium nitrate, pH 5.0, was added toprevent coagulation, and the plasma was stored at 20° C. The normalconcentration of hFIX in human plasma is 3 μg/ml, Approximately 15 ng/ml(72 days after transfection) to 1050 ng/ml (48 days after transfection)of active human factor IX were produced in individual animals injectedwith the DNA complex (Table 102). It is not clear if the smallvariations in the concentration of recombinant hFIX found in the animalsrepresent a difference in delivery efficiency or in the expression ofthe newly introduced gene. The hFIX gene was expressed in the animalsfor up to 140 days (the duration of the experiment), with the highestlevel noted at 48 days (Table 102).

It has been established using transgenic animals (McGrane, et al., 1988,1990; Short, et al. 1992) that transcription from the PEPCK promoter canbe induced by the administration of a high protein-low carbohydratediet. In order to demonstrate the regulated expression of the transgene,we analyzed the blood of transfected animals for the presence of hFIX byWestern blot hybridization before and after feeding a high protein-lowcarbohydrate or a normal chow diet for 1 week. We noted up to 3-foldinduction of PFIX gene expression in animals containing the PFIX genefor up to 140 days after injection of the DNA complex. The samePEPCK-hFIX gene, introduced into the livers of rats using an alternativemethod of receptor-mediated gene transfer targeting the ASGE, was activefor only two days (Ferkol, et al., 1993); this suggests that the use ofa highly compacted DNA complex may be responsible for the prolongedexpression of the transgene noted in the present study.

Detection of maintained levels of hFIX protein at time points as long as140 days is evidence for expression throughout the experimental timecourse. A human FIX 800 bp. specific transcript was detected by PCRamplification of cDNA generated from total cellular RNA by reversetranscriptase, in the livers of animals expressing functional hFIXprotein (FIG. 3A). The presence of mRNA along the experimentaltime-course would indicate that there is a maintained pool oftranscriptionally active DNA in these animals which persistence willexplain the prolonged expression and detection of hFIX and specificmRNA.

We have also established the presence of the transfected DNA in theliver of animals 32 days after transfection, and investigated itsphysical state. The DNA extracted was subjected to restriction enzymeanalysis with Bgl II that linearizes the plasmid (4.5. Kb) and with EcoRI that releases the 2.6 Kb chimeric gene from the plasmid. Southern blothybridization using a hFIX specific probe demonstrated that thetransfected DNA remains in episomal state in the transfected livers,since BglII produced a single band consistent with the size of thelinear plasmid in contrast to the expected smeared hybridization whenrandom integration occurs (FIG. 3B). We cannot rule out the possibilitythat a small proportion of the transfected DNA may have undergone randomintegration into the enome of the transfected animals. However, webelieve that this event is improbable since the liver has not beensubjected to stimulation of mitosis (i.e., partial hepatectomy).

The asialoglycoprotein receptor is present only in parenchymal cells ofthe liver. Nevertheless, it has been shown that asialoglycoproteins andother galactose terminal ligands can be taken up by macrophages by amechanism dependent on the size of the molecular ligand. SeeSchlepper-Schafer, J. et al., Exp. Cell. Res. 165:494 (1986);Bijsterbosch, M. K., et al., Mol. Pharmacol 36:484 (1989); andBijsterbosch, M. K., et al., Mol. Pharmacol 41:404 (1992). The size ofthe DNA/ligand-poly-L-lysine complex in our experiments would becompatible with the discriminating range of the asialoglycoproteinreceptor. In order to investigate the specificity of the DNA complex wehave obtained DNA from different tissues in a transfected animal andamplified the transfected DNA by PCR. Our results show the absence ofamplifiable DNA in tissues other than liver, which would indicatespecific uptake by hepatocytes. It is especially interesting that thereis no detectable uptake in macrophage-containing tissues like lung andspleen. In contrast, we have detected transfected DNA in the lung andspleen of animals transfected using the method described by Wu, et al.for receptor-mediated endocytosis by means of the asialoglycoproteinreceptor. We believe that the small size of the molecular ligandachieved in our experiments is responsible for the specificity of uptakereported here.

EXAMPLE 2

In this Example a different promoter-gene construct (SV40/luciferase) isdelivered to a different cell type (macrophages) by means of a differenttarget cell binding moiety.

Introduction

The recognition and uptake of circulating glycoproteins by specificcells are determined by the nature of the exposed sugar residues presenton the surface of the molecule. The clearance systems of specificglycoproteins are relatively exclusive and are mediated by specifictypes of cells. The mannose receptor recognizes glycoproteins withmannose, glucose, fucose, and N-acetylglucosamine residues in exposed,non-reducing positions. Various proteins and glycoprotein conjugatesbearing these carbohydrate residues bind to isolated alveolarmacrophages, and mannose-terminal glycoproteins infused into thecirculation of rats are cleared by Kupffer cells in vivo. Conversely,galactose-terminal glycoproteins, which are cleared by theasialoglycoprotein receptor on hepatocytes, are not recognized by thesecells. This cell-surface receptor is expressed by a variety ofmacrophage subtypes but not circulating monocytes, and mediates thedelivery and internalization of mannose-terminal glycoproteins. Themannose receptor recycles constituitively from a pre-lysosomalcompartment to the cell surface, and receptor expression is regulated bymacrophages.

Macrophages present in various organs (i.e. liver, spleen, lung, andbone marrow) which bind mannose-terminal glycoproteins and therefore maybe a target cell for receptor-mediated gene transfer. We tested thishypothesis by examining our ability to deliver functional exogenousgenes cells which express the mannose receptor. In this report, amannose-terminal neoglycoprotein carrier was synthesized and employed asa ligand for receptor-mediated gene transfer to primary murinemacrophages isolated from the peritoneal exudates, which abundantlyexpress the receptor on their surface. In addition, the reporter geneswere transferred successfully into macrophages present in the liver andspleen of intact rats using the mannose-terminal neoglycoproteincarrier.

Methods

Materials: DNA-modifying enzymes, nucleotides, and5-Bromo-4-chloro-3-indolyl-β-D-galactopyranoside were purchased fromBoehringer Mannheim (Indianapolis, Indiana, USA). All chemicals,including poly (L-lysine), a-D-mannopyranosylphenyl isothiocyanatealbumin, and a-D-galactopyranosylphenyl isothiocyanate, were obtainedfrom the Sigma Chemical Company (St. Louis, Mo., USA). Luciferase assaysystem was obtained from Promega (Madison, Wis., USA). The rabbitanti-β-galactosidase antibody and fluorescein isothiocyanate-conjugatedgoat anti-rabbit IgG was obtained from the 5 Prime to 3 Prime, Inc. Allmedia, sera, and antibiotics were obtained from Gibco Laboratories(Grand Island, N.Y., USA).

Preparation of mannose-terminal glycoprotein carrier-Syntheticglycoprotein carriers were constructed in which poly (L-lysine), averagechain length 100 (M_(r) 20,000 Da), was glycosylated usinga-D-mannopyranosyl phenylisothiocyanate dissolved inN,N-dimethylformamide. The solution was adjusted to pH 9.5 by theaddition of 1M Sodium carbonate, pH 9.5. Shielded from light andincubated for 16 hours at 22° C., the solution was dialyzed against 5 mMSodium chloride for two days. Approximately 0.8 to 1.0% of the amineside chains in the polylysine are glycosylated, as determined byabsorbance spectroscopy at 250 nm. As a control, an alternativeglycoprotein carrier was synthesized by substituting a-D-mannopyranosylphenylisothiocyanate with a-D-galactopyranosyl phenylisothiocyanate.

Reporter genes and plasmid preparation: The expression plasmid pGEMluccontained the SV40 viral promoter and enhancer elements ligated to theP. pyralis luciferase gene. The plasmids PCMVZ and pCMVIL2r, consistingof the cytomegalovirus (CMV) promoter linked to the E. coli lacZ and theinterleukin 2 receptor genes, respectively, were also used as reportergenes. The plasmids were grown in E. coli DH5a, extracted, and purifiedby standard techniques (14). Digestions of the plasmids with restrictionendonucleases yielded the appropriate size fragments, and purity wasestablished by 1.0% agarose gel electrophoresis. The sizes of plasmidsare as follows: pGEMluc , 6.0; pCMVlacZ, 10.9; and pCMVIL2r, 5.4 kB. Nobacterial genomic DNA was present in the plasmid preparations.

Preparation of mannose-terminal glycoprotein carrier-DNA complexes.Complexes were formed analogously to Example 1, however, the DNA wasabout 80% supercoiled and 20% open circular.

Cells and cell culture. Primary macrophages were isolated from theperitoneal cavity of mice four days after the intraperitoneal injectionof one milliliter of Brewer's thioglycolate medium. The macrophages fromthe peritoneal exudate were collected as previously described, andmaintained in RPMI Media 1640. This method yielded approximately 5×10⁶cells per mouse, of which 40-75% were mononuclear phagocytes based onmorphological characteristics of the cells and cytochemicalidentification. Transfections were performed one or two days aftercollection. The isolated cells were approximately 30-60% confluent atthe time of transfection. Viability of cells was determined by serialcell counts and trypan blue exclusion.

DNA delivery to macrophages in culture: One day after isolation, thecells isolated from the peritoneal exudates of mice were washed oncewith PBS (pH 7.4) and the media was changed immediately beforetransfection. The conjugate-DNA complex, containing 5 μg (0.4-0.7 pmol)plasmid, was applied to the culture medium and permitted to remain onthe cells for 24 hours unless the experiment dictated otherwise. Thecells were then either harvested for protein extraction or fixed for insitu β-galactosidase assays at several timepoints after transfection.

Animals: Adult, male Sprague-Dawley rats, weighing approximately 250 g.,were anesthetized with ether. Using aseptic technique, 0.3 to 0.6 ml ofa solution containing 300 μg (20.8-42.0 pmol) of an expression plasmidcomplexed to the carrier was injected into the caudal vena cava. Therats were killed at different intervals after infusion of the complexesand the livers, lungs, and spleens of transfected animals were removedfor analysis. Furthermore, macrophages were isolated from the alveoli,the bone marrow, and spleen. Bone marrow cells were obtained from therat's femur. The femur was surgically removed after the experimentalanimal was sacrificed, and one milliliter of media was infused into andaspirated from the marrow cavity. A single-cell suspension of the marrowwas prepared by gently aspirating the cells with a Pasteur pipette. Thecells extracted from the bone marrow were maintained in RPMI Media 1640for 8-12 hours and permitted to attach to glass slides, at which timethe adherent cells were fixed for immunocytochemical staining.Non-transfected and mock transfected animals were used as controls inall analyses. The animal research protocol was reviewed and approved bythe Case Western Reserve University Institutional Animal Care Committee.

Cytochemical assay for β-galactosidase activity: Individual cellsexpressing β-galactosidase were identified following incubation with5-Bromo-4-chloro-3-indolyl-β-galactopyranoside (X-gal) as describedpreviously. Briefly, the cells were fixed with a solution of itglutaraldehyde in PBS for 15 minutes, and then incubated with a solutioncontaining 0.5% X-gal for 12 hours at 37° C. The cells were also stainedfor nonspecific esterase activity, which produces an insolublegrey-black dye. A minimum of 100 cells in tissue culture were counted todetermine the percentage of cells expressing β-galactosidase.

Individual cells expressing β-galactosidase in tissues were identifiedfollowing incubation with X-gal as described previously. Briefly, thecells were fixed with a solution of 0.5% glutaraldehyde in PBS for 10minutes, washed twice with PBS, pH 7.5, and then incubated with asolution containing 0.5% X-gal, 5 mM Potassium ferricyanate, 5 mMPotassium ferrocyanate, and 1 mM Magnesium chloride inphosphate-buffered saline (pH 7.4) for 6 hours at 37° C. The stainedtissues were fixed in 2% paraformaldehyde/0.5% glutaraldehyde in PBSovernight at 4° C., paraffin embedded by standard procedure, and cutinto 5 μm sections. The sections were counterstained with 0.1% nuclearfast red. The adjacent tissue sections were also stained for nonspecificesterase activity, which appears brown-black. Blue colored cells wereidentified by light microscopy.

Cytochemical identification of macrophages. Cells and tissue sectionswere stained nonspecific esterase activity, which is relatively specificfor mononuclear phagocytes. The cell smears were fixed as describedabove, and incubated with a filtered solution containing a-naphthylacetate and Fast Blue BB salt for 10 minutes at room temperature. Tissuesections were stained with this solution for 1-3 hours, andcounterstained with 0.1% nuclear fast red.

Immunocytochemical staining for beta-galactosidase: The expression ofthe transgene in cells isolated from tissues (spleen and bone marrow)transfected in vivo with the plasmid PCMVZ was determined by indirectimmunofluorescence. Cell smears were fixed with methanol/acetone for 2minutes at room temperature, and the cells were incubated with a rabbitanti-b-galactosidase polyclonal antibody for one hour at 37° C. Theprimary antibody was diluted 1:100 in PBS for immunodetection in thefixed cell smears. Fluorescein isothiocyanate conjugated anti-rabbitimmunoglobulin G diluted 1:100 in PBS was used as the secondaryantibody. The cells were also counterstained with propidium iodide,which produces red fluorescence in the cell nucleus. Between eachincubation, the cells were washed three times for five minutes with PBS.The stained cells were examined by fluorescent microscopy.

Assays for luciferase activity: Cells in culture were harvested, lysed,and analyzed for luciferase activity as described previously. Tissueswere harvested from transfected and control rats after the animals weresacrificed and perfused in situ with 50 milliliters of cold PBS, pH 7.5.The tissues were homogenized in lysis buffer and permitted to incubateat 22° C. for 10 minutes. The cell lysates were subsequently centrifugedfor 5 minutes at 4° C., and the protein extracts were analyzed forluciferase activity. The lysates were assayed for protein content andthe measured integrated light units were standardized for total proteincontent. All measurements were performed in triplicate and expressed asan average of the values.

Statistical analysis: Data are expressed as means +standard error of themean (SEM), and evaluated by an analysis of variance using theStudent-Newman-Keuls (SNK) test.

Results

In vitro Transfection of Primary Macrophages using the Mannose-terminalGlycoprotein Carrier

Using an expression plasmid (PCMVZ) encoding the E. coli lacZ gene as areporter gene, complexes of the plasmid and the mannose-terminalglycoprotein carrier were applied to cells peritoneal exudates cellsisolated from mice. Twenty-four hours after transfection, the cells wereexamined for β-galactosidase activity. The number of transfected cellsvaried from 5 to 26 per cent of all cells examined. In addition, theproportion of cells with non-specific esterase activity, a cytochemicalmarker characteristic of monocytes and macrophages, that expressed thetransgene ranged from 40% to 75%. Transfections using complexesconsisting of an irrelevant plasmid (pGEMluc) bound to the carrier orthe expression plasmid (pCMVZ) bound to a galactose-terminalglycoprotein carrier no significant β-galactosidase activity in theexudate cells. Faint blue staining was noted in these control cells,which was most likely due to endogenous β-galactosidase activity.Nevertheless, the percentage and intensity of blue stained cells in thecontrols was markedly less than that in the transfected dishes, Themannose-terminal glycoprotein carrier-DNA complex appeared to benon-toxic to cells since the percentage of cells viable, based on cellcounts and trypan blue staining, after treatment was not significantlydifferent than controls.

Complexes of the mannose-terminal glycoprotein carrier and theexpression plasmid pGEMluc were applied to cells isolated fromperitoneal exudates for increasing periods of time, and luciferaseactivity was measured in protein extracts of the transfected cells 24hours following transfection. As noted in the previous experiments, thelevel of expression of the transferred gene varied. An eight-foldincrease in relative luciferase activity in transfected cells waspresent (p<0.01), whereas protein extracts obtained from cells treatedwith a complexes formed using a galactose-terminal glycoprotein carrierdid not express activity significantly different than thenon-transfected control. Furthermore, the addition of a one hundred-foldmolar excess of mannosylated bovine serum albumin over complex to theculture media immediately before transfection, which should compete withthe carrier for the mannose receptor, completely inhibited the uptakeand expression of the reporter gene (p<0.01). The duration of thetransgene expression in these cells was also examined. The complexes ofthe mannose-terminal glycoprotein carrier and the expression plasmidpGEMluc were applied to cells for 24 hours, and protein extracts wereassayed for luciferase activity at several timepoints aftertransfection. Optimal transgene expression was detected one day aftertreatment, and luciferase activity decreased to control levels eightdays post transfection.

In vivo Transfection of Macrophages using the Mannose-terminalGlycoprotein Carrier

The mannose-terminal glycoprotein carrier was used to transfer reportergenes into the spleen and livers of intact animals. Rats wereanesthetized, and 300 μg of plasmid (pGEMluc) was complexed to themannose-terminal glycoprotein carrier and infused slowly into the caudalvena cava over several minutes. Control and mock transfections ofanimals using complexes consisting of an irrelevant plasmid (pCMVlacZ)bound to the carrier were also performed in parallel. All animalsinjected with the complex survived. Luciferase assays were performedfour days after infusion of the complexes in tissue homogenatesextracted from liver, lungs, and spleen. We observed significant levelsof transgene expression in the protein extracts from the spleen obtainedfrom transfected animals. Lower levels of luciferase activity was foundin the liver and lung. Non-transfected rats and animals treated with thecomplexes consisting of an irrelevant plasmid (pCMVlacZ) bound to themannose-terminal glycoprotein carrier had no significant luciferaseactivity in protein extracts from any tissue. Twelve days aftertransfection, luciferase activity approximated background levels in alltissues examined.

The cellular distribution of the transgene expression was examined insections of spleen and liver three days after the injection of complexescontaining pCMVlacZ. The tissues were analyzed for b-galactosidaseactivity by a cytochemical stain. An animal treated with complexes madeusing an irrelevant plasmid (pCMVIL2r) served as control.Beta-galactosidase expression was detected in several small cells in thespleen located in the subcapsular region, which conformed to thedistribution of cells that expressed nonspecific esterase activity basedon cytochemical staining. No beta-galactosidase activity was found inthe corresponding cells of the control spleen. Rare, blue-stained cellswere present in hepatic sections of the transfected animal, and nohepatic endothelial cells, which also have surface mannose receptors,expressed the transgene. Nucleated cells were also isolated from thespleen and stained in vitro. Furthermore, cells extracted from the bonemarrow and bronchoalveolar lavage fluid of the transfected and controlanimals were also treated with a solution containing X-gal and examinedfor beta-galactosidase activity. Approximately 10-20 percent of thenucleated cells obtained from the spleen stained blue. Rare cells fromthe mock transfected animal were also faintly blue stained, most likelydue to an endogenous β-galactosidase. Nevertheless, the percentage andintensity of blue stained cells in the controls was significantly lessthan that found in the control animal.

A polyclonal antibody directed against the bacterial beta-galactosidasewas used for the immunocytochemical localization of the transgeneproduct to establish that the blue-stained cells in the spleen are notdue to endogenous beta-galactosidase or the nonspecific hydrolysis ofX-gal. Nucleated cells isolated from the spleen and bone marrow of theanimals described above were stained with antibody directed againstbeta-galactosidase and fluorescein isothiocyanate conjugated anti-rabbitand examined for immunofluorescence. A number of the isolated cells,which were morphologically similar to the blue stained cellsdemonstrated in the cytochemical assay, had immunofluorescent staining.In addition, these cells had nonspecific esterase activity.

Discussion

We have developed a synthetic glycoprotein complex capable of mediatingtransfer of functional genes into macrophages in culture and the liversof whole animals. Expression plasmids noncovalently bound to anmannose-terminal glycoprotein carrier can be introduced efficiently intocells that express the mannose receptor. The delivery of DNA by areceptor-mediated gene transfer system is dependent on the presence ofreceptors on the surface of the targeted cell. Cells that fail toexpress the asialoglycoprotein receptor were not transfected by thissystem. In addition to macrophages, other cell types present in theperitoneal exudate that fail to express the mannose receptor, i.e.,granulocytes, lymphocytes and fibroblasts, were not transfected. Theexpression of the reporter gene was localized to cells that had eithernon-specific esterase or peroxidase activity, reliable cytochemicalmarkers used for macrophage identification.

The specificity and affinity of the ligand for the specific receptor areof considerable importance for the delivery of exogenous genes.Macrophages bind mannose-terminal glycoproteins with high affinity andspecificity. The mannose-terminal glycoprotein carrier successfullyintroduced reporter genes into macrophages in culture and in intactanimals, whereas transgene expression was not detected in cellstransfected using a galactose-terminal glycoprotein carrier. Uptake doesnot appear to be due to a non-specific increase in pinocytosis orphagocytosis secondary to the presence glycoprotein in the culturemedium. The delivery and expression of the plasmid is inhibited by theaddition of mannosylated bovine serum albumin to the culture medium,which presumably competes for the binding site(s) on the mannosereceptor. Finally, the substitution of an alternative monosaccharide formannose could increase the affinity of the DNA-carrier complex, sincethe mannose receptor also recognizes glycoproteins with glucose, fucose,and N-acetylglucosamine residues in exposed positions. In addition, genetransfer efficiency could potentially be improved by altering thecarbohydrate residue to an oligosaccharide, i.e. oligomannose, sincemonosaccharides are poorer ligands for the receptor than are polyvalentglycoproteins.

A major factor in determining the level of expression of the genestransferred into target cells involves the survival and delivery of theexogenous DNA to the nucleus. Expression of genes introduced byreceptor-mediated mechanisms may be limited by the trapping anddegradation of the complex in endosomal compartments. Mannose-terminalglycoproteins are introduced into macrophages by receptor-mediatedendocytosis, delivered to a pre-lysosomal acidic compartment, andsubsequently trafficked to the secondary lysosomes. Apparently, aportion of the introduced conjugate avoids destruction since thetransferred DNA must escape degradation after the complex has enteredthe cell in order for the transgene to be expressed. The physical stateof the DNA transferred into cells by these delivery systems may alsocontribute to its survival and subsequent expression, and highly compactform of DNA may be more resistant to nuclease digestion. Furthermore,the small size of the carrier-DNA complex may also permit theintroduction of the plasmid into the cells of the reticuloendothelialsystem specifically via the mannose receptor and not by phagocytosis.

This study illustrates the potential of specifically directing genetransfer into macrophages by targeting the mannose receptor, andtheoretically could provide an approach to the treatment of variousinborn errors of metabolism, like Gaucher disease. Pharmacologictherapies that also target the mannose receptor have been shown to beeffective in patients with Gaucher disease. Repeated treatments ofaffected individuals with modified human glucocerebrosidase, in whichthe outer carbohydrate moieties are cleaved to expose terminal mannoseresidues, have had substantial clinical improvement in their disease, asdemonstrated by reduction in hepatosplenomegaly and resolution ofanemia. Unfortunately, the cost of this therapy has been prohibitive tomany patients. Bone marrow transplantation has been shown to be curativein the non-neuropathic form of the disease, yet the potentialcomplications of transplantation precludes this procedure in manypatients, particularly those in individuals with mild disease. However,because Gaucher disease can be corrected by bone marrow transplantation,one potential approach that has been proposed for the gene therapy ofGaucher disease involves the ex vivo transfer of the normalglucocerebrosidase gene into autologous hematopoietic stem cells andtheir subsequent introduction into the patient. Alternatively,lymphoblasts could be harvested from the affected individual, infectedwith replication-incompetent, recombinant retrovirus containing thewild-type gene, and returned to the patient. The secreted enzyme wouldenter the macrophages via the mannose receptor, thus becoming thesecondary targets of therapy. In the system we describe in thismanuscript, the macrophage would be the primary target for geneticcorrection. Practical questions regarding the efficiency of genedelivery, duration and level of expression achieved using thistechnique, and the immunologic properties of the DNA-carrier complexesneed to be addressed. Nevertheless, receptor-mediated gene therapy hasthe potential of providing a non-invasive approach to the treatment ofsuch diseases.

EXAMPLE 3

We have also used a Fab fragment of a monoclonal antibody directedagainst the rat polymeric immunoglobulin receptor that is expressed inthe airway epithelia. The Fab peptide was covalently coupled topoly-L-lysine and complexed to an SV40-luciferase expression vectorusing the procedure described below. Rats injected with the DNA complexhad luciferase activity for as long as 8 days (the duration of theexperiment) only in tissues that expressed the receptor. These findingunderline the flexibility of this system for delivering DNA to specifictissues of an adult animal. Introduction

Several methods of gene transfer into the respiratory tract have beendeveloped that permits the introduction of functional genes into cellsin vivo. However, many of these approaches have lacked specificity andare cytotoxic. Replication deficient, recombinant adenoviruses have beenused to deliver the reporter genes to respiratory epithelial cells in avariety of animal models. However, the physiologic effects of treatmentwith adenovirus are not well understood, and recent-evidence suggeststhat the first-generation adenoviral vectors administered at high viraltiters to animals produce a substantial inflammatory response in thelung. Liposomes have also been used to transfer functional genes to theairway epithelium, but this approach has generally been toxic to cellsand lack specificity.

Receptor-mediated gene transfer may provide a method for delivering DNAto specific target cells using a non-infectious, non-toxic vector. Thisform of gene transfer allows specific tissue targeting with DNA plasmidsof considerable size, allowing for delivery of not only the transgene,but also promoter and enhancer elements. In the case ofreceptor-mediated systems, the delivery of exogenous DNA is dependent onthe stability of the DNA-carrier complex, the presence and number ofspecific receptors on the surface of the targeted cell, thereceptor-ligand affinity and interaction, and efficient internalizationof the complex. Furthermore, expression of the transferred genes rely ontheir escape from the endosomal vesicles and trafficking to the targetcell's nucleus. The duration of transgene expression in whole animalsdelivered by exploiting receptor-mediated endocytosis has been generallybeen transient, returning to background levels within seventy-two hoursafter treatment. This has been the case for reporter genes introducedinto airway epithelial cells via the intratracheal route usingadenovirus-polylysine and transferrin-adenovirus-polylysine vectors.

We have demonstrated that in primary cultures of human trachealepithelial cells, targeting the polymeric immunoglobulin receptor (pIgR)permits the efficient delivery of the transgene specifically to cellsthat bear the receptor. The polymeric immunoglobulin receptor isexpressed only in mucosal epithelial cells, including airway epithelialand submucosal gland cells, and is specifically adapted for theinternalization and nondegradative transfer of large molecules. In thisreport, we show that targeting the polymeric immunoglobulin receptor invivo results in expression of the transgene in tissues that containreceptor-bearing cells which was maximal six days after transfection.

Methodology

Materials. DNA-modifying enzymes, nucleotides, and5-Bromo-4-chloro-3-indolyl-β-D-galactopyranoside were purchased fromBoehringer Mannheim (Indianapolis, Ind., USA). Luciferase assay systemwas obtained from Promega (Madison, Wis., USA). Protein A MAPS agarosecolumns were purchased from BioRad (Richmond, Calif., USA). Papain andpoly (L-lysine) were obtained from Sigma Chemical Company (St. Louis,Mo., USA), and N-Succinimidyl-3-(2-pyridyldithio)proprionate was fromPierce Chemical Company (Rockford, Ill., USA). The mouse monoclonalanti-human interleukin 2 receptor antibody was obtained from DakoCorporation. (Carpenteria, Calif., USA), and the fluoresceinisothiocyanate-labelled secondary goat anti-mouse antibody was fromSigma Immunochemicals (St. Louis, Mo., USA). The Vectastain ABC method,used in the immunoperoxidase staining procedure, was purchased fromVector Laboratories (Burlingame, Calif., USA). All media, sera, andantibiotics were obtained from Gibco Laboratories (Grand Island, N.Y.,USA).

Preparation of Fab fragments. The isolation and papain digestion ofantibodies derived from rabbits immunized with rat secretory componenthas been described previously. Briefly, polyclonal antibody was isolatedfrom rabbit serum using a Protein A MAPS agarose column as described bythe manufacturer. Isolated immunoglobulin G (2 mg) was treated with 20μg papain for 12 hours at 37° C. in the presence of loo mM sodiumacetate (pH 5.5) 50 mM cysteine, and 1 mM EDTA. The Fab fragment wasseparated from intact antibody and Fc fragments by Protein Achromatography. An irrelevant Fab (IFab) was generated by papaindigestion of IgG from pre-immune rabbit serum.

Preparation of Fab-polylysine conjugates. The Fab fragment of theanti-pIgR immunoglobulin G was covalently linked to poly (L-lysine)(M_(r) 10,000 Da) using the heterobifunctional crosslinking reagentN-Succinimidyl 3-(2-pyridyldithio) proprionate (SPDP). The Fab fragmentwas incubated with a seventy-five fold molar excess of SPDP in 0.1Mphosphate buffered saline (PBS), pH 7.5, at 22° C. for 60 minutes. Afterintroduction of 2-pyridyl disulfide structures onto the Fab fragment,unreacted SPDP and low molecular weight reaction products were removedby dialysis. The disulfide bridges of the modified Fab fragment werecleaved with 25 mM dithiothreitol. Both the poly (L-lysine) and SPDP wasadded in fifteen fold molar excess to the modified Fab fragment, and thereaction was carried out at 22° C. for 24 hours. The conjugate wasdialyzed to remove low molecular weight reaction products, and analyzedby separating the resultant proteins on a 0.1% SDS-7.5% polyacrylamidegel electrophoresis. As described previously, analysis of the conjugatedemonstrated a protein that migrated slowly, corresponding to a proteingreater than 200 kDa in size.

Reporter genes and plasmid preparation. The expression plasmid pGEMluccontained the SV40 viral promoter ligated to the P. pyralis luciferasegene. The plasmids pCMVZ and pCMVIL2r, consisting of the cytomegalovirus(CMV) promoter linked to the E. coli lacZ and the interleukin 2 receptorgenes, respectively, were also used as reporter genes. For studies ofluciferase activity, these plasmids were employed as irrelevant DNA(IDNA) controls. The plasmids were grown in E. coli DH5a, extracted, andpurified by standard techniques. Digestions of the plasmids withrestriction endonucleases yielded the appropriate size fragments, andpurity was established by 1.0% agarose gel electrophoresis. The sizes ofplasmids are as follows: pGEMluc , 6.0; pCMVlacZ, 10.9; and pCMVIL2r,5.4 kB. No contamination with bacterial genomic DNA or RNA was presentin the plasmid preparations.

Preparation of Fab-polylysine-DNA complexes. The carrier-DNA complexeswere formed using a method described previously.

Animals: The anti-rat secretory component Fab antibody-polylysinecarrier was used to transfer reporter genes into the airways and liversof intact animals. Adult, male Sprague-Dawley rats, weighingapproximately 250 g., were anesthetized. Using aseptic technique, 0.3 to0.6 ml of a solution containing 300 μg of an expression plasmidcomplexed to the carrier was injected into the caudal vena cava. Therats were sacrificed at several different times after infusion of thecomplexes and various organs were removed for analysis. Mocktransfections of animals using complexes consisting of an irrelevantplasmid bound to the carrier or the expression plasmid bound to acarrier made with an irrelevant Fab fragment were also performed inparallel. The animal research protocol was reviewed and approved by theCase Western Reserve University Institutional Animal Care Committee.

Cytochemical assay for β-galactosidase activity: Individual cellsexpressing β-galactosidase in tissues were identified followingincubation with 5-Bromo-4-chloro-3-indolyl-β-galactopyranoside (X-gal)as described previously. Briefly, the cells were fixed with a solutionof 0.5% glutaraldehyde in PBS for 10 minutes, washed twice with PBS, pH7.5, and then incubated with a solution containing 0.5% X-gal, 5 mMPotassium ferricyanate, 5 mM Potassium ferrocyanate, and 1 nM Magnesiumchloride in phosphate-buffered saline (pH 7.4) for 4 hours at 37° C. Thestained tissues were fixed in 2% paraformaldehyde/0.5% glutaraldehyde inPBS overnight at 4° C., paraffin embedded by standard procedure, and cutinto 5 μm sections. The sections were counterstained with nuclear fastred. Blue colored cells were identified by light microscopy. A minimumof 100 cells were counted to determine the percentage of cells persection that express β-galactosidase. In addition, adjacent sectionswere stained with Alcian blue/periodic acid Schiff or haematoxylon/eosinusing standard protocols.

Assays for luciferase activity: Cells in culture were harvested, lysed,and analyzed for luciferase activity as described previously. Tissueswere harvested from transfected and control rats after the animals weresacrificed and perfused in situ with cold PBS, pH 7.5, for five minutes.The tissues were homogenized in lysis buffer and permitted to incubateat 22° C. for 10 minutes. The cell lysates were subsequently centrifugedfor 5 minutes at 4° C., and the protein extracts were analyzed forluciferase activity. The lysates were assayed for protein content andthe measured integrated light units (10 second interval) werestandardized for total protein content. All measurements were performedin triplicate and expressed as an average of the values.

Immunohistochemical staining for the interleukin 2 receptor. Theexpression of the transgene in tissues transfected with the plasmidpCMVZ was determined by indirect immunofluorescence. Frozen sections ofvarious tissues were fixed with acetone for 10 minutes at -20° C., andtreated with for ten minutes at 22° C. to reduce autofluorescence. Thesections were then incubated with 10% goat serum in PBS, pH 7.5, for onehour at room temperature. The cells were incubated sequentially with amouse monoclonal anti-interleukin 2 receptor antibody and fluoresceinisothiocyanate-conjugated goat anti-mouse IgG. Both antibodies werediluted 1:100 in PBS, and between each incubation, the cells were washedthree times for five minutes with PBS, pH 7.5. The stained cells wereexamined by fluorescent microscopy.

Results

In vivo Transfection using the Anti-Secretory Component FabAntibody-Polylysine Carrier

All animals injected with the anti-rat secretory component Fabantibody-polylysine carrier-DNA complex survived. Luciferase assays wereperformed 48 hours after infusion of the complexes in tissue homogenatesextracted from liver, lungs, spleen, and heart. We observed significantlevels of transgene expression in the protein extracts from the liverand lungs obtained from transfected animals. No detectable luciferaseactivity was found in the spleen and heart, tissues that do not expressthe pIgR. Furthermore, animals treated with the complexes consisting ofan irrelevant plasmid (pCMVlacZ) bound to the carrier or the expressionplasmid (pGEMluc) bound to a carrier based on an irrelevant Fab fragmentresulted in no significant luciferase activity in any tissue examined.Thus, only tissues that contain cells bearing pIgR are transfected, andtransfection cannot be attributed to the nonspecific uptake of anirrelevant Fab antibody-based complex.

A time course of the expression of the transferred gene, in whichluciferase activity in protein extracts derived from the four tissueswas measured at different timepoints after injection of the complex, wasdeveloped. Luciferase activity persisted in the liver and lung, tissueswhich have pIgR, achieving maximum values of 13795±4431 and461402±230078 integrated light units (ILU) per milligram of proteinextract, respectively, at four to six days after injection. Tissues thatfailed to express the receptor did not have significant transgeneexpression.

The cellular distribution of the transgene expression was examined insections of various tissues. Three days after the injection of complexescontaining pCMVlacZ, tissue sections of trachea, lung, and liverunderwent cytochemical staining for b-galactosidase activity. An animaltreated with complexes made using an irrelevant plasmid (pCMVIL2r)served as control. Expression in the trachea was limited to the cellslining the epithelial surface. No beta-galactosidase activity wasdetected in the tracheal sections from the mock transfected animal. Theexpression of the transgene was variable, and in some areas of therespiratory epithelium greater than 50% of the cells stained blue. Ingeneral, expression ranged from 10-20% of the tracheal epithelial cells.Both ciliated and secretory (goblet) respiratory epithelial cellsexpressed beta-galactosidase activity, based on Alcian blue/periodicacid Schiff staining of adjacent sections of the airway. No expressionfrom the transgene was detected in the terminal airways or alveoli ineither the transfected or control animal (data not shown). This conformsto the distribution of epithelial cells that express the pIgR based onin situ immunohistochemical staining. Rare submucosal glands wereevident in the tracheal sections, and only faint blue staining wasnoted. No inflammatory response was found in any of the trachealsections from the non-, mock-, and transfected animals. In addition, amouse monoclonal antibody directed against the human interleukin 2receptor, a surface protein that has been used as a reporter in thetransduction of respiratory epithelial cells in vitro but is notnaturally expressed in these cells, was used for immunofluorescentlocalization of the transgene product in the trachea of the animaltransfected with the plasmid pCMVIL2r. Serial sections of the tracheawere examined for the presence of fluorescence, and the apical membraneof numerous respiratory epithelial cells from the transfected animalstained appropriately. No specific fluorescent staining was detected inthe airway epithelia of an animal mock-transfected with pCMVlacZ. Rare,blue-stained hepatocytes were also found in hepatic sections of thetransfected animal. Transgene expression was not identified in thelivers from either non- or mock-transfected rats.

Discussion

We report the successful transfer of reporter genes into the airwayepithelium in vivo following the injection of a targeting complexconsisting of the Fab portion of immunoglobulin G directed against therat polymeric immunoglobulin receptor conjugated to poly (L-lysine), andnoncovalently bound to plasmid DNA. This technique specificallydelivered the transgene to the liver and lung, tissues in which thisreceptor is expressed. Other tissues that do not express the receptor,like the spleen and heart, were not transfected. In addition, followinginjection of a conjugate prepared with irrelevant Fab fragments noexpression was detected, and a complex prepared with a plasmidcontaining an irrelevant reporter gene also failed to produce detectableluciferase activity. Thus, this complex specifically targetsreceptor-bearing tissues and the normal trafficking of the receptor'snatural ligands does not interfere with the uptake of the transgene invivo.

Most of the strategies for gene transfer into the respiratory tractcurrently available depend on viral vectors which do not specificallytarget respiratory epithelial cells, and rely upon the intratrachealroute of delivery to permit targeting of the airway. Intratrachealinstillation has also been used to specifically direct gene transfer byother means, like liposomes and adenovirus-transferrin-polylysineconjugates, to the airway epithelium. Systemic delivery of DNA bound tocationic liposomes has not been selective and transfers functional genesto a number of cell types in different tissues. The specificity ofreceptor-mediated gene transfer for cells that bear the pIgR may beuseful in targeting defective cells in the airways of patients withcystic fibrosis.

EXAMPLE 4

INTRODUCTION

Familial hypercholesterolemia (FH) is a human genetic diseasecharacterized by fulminant atherosclerosis and cardiovascular disease. Amutation in the gene for the receptor that mediates the uptake of thelow density lipoprotein (LDL) is responsible for this disease. One inevery 500 people is heterozygote for a mutation in the LDL receptor genethat is responsible for FH. As a result, LDL is removed from theirplasma at only two thirds the normal rate. In the fourth to fifth decadeof life, the elevated levels of LDL in plasma cause symptomaticatherosclerosis in these patients. FH-homozygotes (one in a millionpeople) have little or no functional LDL receptor, depending on thedomain of the protein that is affected by the mutation. This results insymptomatic coronary atherosclerosis before the age of 20. Treatmentwith bile acid-binding resins and inhibitors of cholesterol synthesishas been considerably successful in heterozygous FH patients bystimulating the production of LDL receptor from the single normal gene.In FH homozygotes there is no response to drug therapy. Because of theabsence of a normal gene that can be stimulated, the replacement of themutated gene is the only possible approach for the treatment ofhomozygous FH patients. Since the liver is the major organ responsiblefor LDL catabolism, the two approaches taken for the treatment of thedisease target this organ: liver transplantation and gene therapy.Transplantation of a normal liver into a patient with FH can correcthyperlipidemia, suggesting that reconstitution of the hepatic LDLreceptor should be sufficient for phenotypic improvement. Based on thisresults, all the approaches undertaken using gene therapy for thetreatment of FH have targeted the hepatocytes.

In order to understand the mechanism of disease, it is necessary to beaware of the metabolism/fate of cholesterol in the organism. Every cellneeds cholesterol for the synthesis of the plasma membrane. The adrenalglands and the corpus luteum in the ovary, in addition, requirecholesterol for the synthesis of steroid hormones. The liver is theorgan with the highest demand because of the production of bile acids.Cholesterol is obtained in peripheral tissues either fromreceptor-mediated uptake of low density lipoproteins (LDL), which arethe main carriers of endogenous cholesterol in the blood, or bybiosynthesis. HMG CoA reductase is the rate-determining enzyme in thepathway. Dietary cholesterol is carried in the bloodstream bychylomicron particles, which are taken up by specific receptors in theliver. In order to provide the different tissues with cholesterol, theliver secretes very low density lipoprotein (VLDL) particles composed oftriglycerides, cholesteryl esters and apoproteins C, E and B-100. Theuptake of triglycerides from VLDL by adipose tissue and muscle convertsthese particles into intermediate density lipoproteins (IDL). The LDLreceptor, present at highest concentration in the liver and adrenalglands but also in the rest of tissues, recognizes the apo E and apoB-100 components of IDL. Thus, under normal conditions IDL is mostlycleared from the bloodstream by LDL receptor-mediated uptake. Theremaining IDL is converted to LDL, which is taken up as well by the LDLreceptor that recognizes the apo B-100 component. The clearance ofcholesterol from the organism is carried out by the liver, where it isconverted to bile acids and secreted into the digestive tract. Althoughmost of the cholesterol is reabsorbed in the terminal ileum for liverreutilization, this pathway provides the route of exit.

Thus, the presence of non-functional LDL receptors that are unable toclear IDL and LDL from the blood results in elevated serum LDL levels,and therefore total serum cholesterol. This is responsible forcholesterol deposition in the artery walls and thus, atherosclerosis.

The Watanabe Heritable Hyperlipidemic (WHHL) rabbit has been previouslyused to study the effectiveness of gene therapy techniques in correctinghypercholesterolemia. A 12 nucleotide in-frame deletion in theligand-binding domain of the LDL receptor, similar to one class ofmutation found in FH patients, results in symptoms, evolution andhistopathology that parallel those of FH.

MATERIALS AND METHODS

Construction of the DNA plasmids

The plasmid DNAs used in this work are pLDLR-17, PCK-hLDLR, PCK-rLDLRand SV40-luciferase. pLDLR-17 was provided by Dr. David Russell(University of Texas, Medical Center, Dallas) and consists of thecytomegalovirus (CMV) promoter/enhancer linked to the human LDL receptorcDNA. It contains a fragment of DNA corresponding to the 5' untranslatedregion (UTR) of the Alfalfa Mosaic Virus 4 (AMV4) RNA linked to thehuman LDL receptor cDNA. This sequence acts as a translational enhancerby decreasing the requirements for initiation factors in proteinsynthesis. The PCK-hLDLR plasmid has been constructed by subcloning thehLDL receptor cDNA from the pLDLR-17 into a pTZ18R vector (Pharmacia)containing the phosphoenolpyruvate carboxykinase (PEPCK) promoter (-460to +73) and an intron and polyadenylation signal from the simian virus40 (SV40) small T antigen. In a two step process, the hLDL receptor cDNAwas excised with SacI and SmaI from the pLDLR-17 and blunted using T4DNA polymerase. The blunted fragment was subcloned into the HincII siteof a pTZ18R vector. The cDNA was then excised with XbaI and SalI andintroduced into the homologous sites of the pTZ18R-PEPCK promoter-SV40polyA plasmid. For the construction of pPCK-rLDLR, the EcoRI-EcoRIfragment from prLDLR-9 (provided by Dr. James Wilson, University ofPennsylvania) containing the rabbit LDL receptor cDNA was subcloned intothe EcoRI site of a pBluescript (Stratagene). This construct wasdigested with SacI and blunted and then digested with XbaI, anddirectionally subcloned into the XbaI-blunted HindIII sites of a pTZ18Rvector containing the PEPCK promoter (-460 to +73) and an intron andpolyadenylation signal from SV40 small T antigen. The SV40-luciferaseplasmid (Promega) contains the SV40 viral promoter and enhancer ligatedto the P. pyralis luciferase gene inserted into the pUC19 vector(Pharmacia).

Formation of the poly-L-lysine-DNA complex

Production of the galactosylated poly-L-Lysine. Poly-L-lysine wasgalactosylated as described (PNAS). Two mg of poly-L-lysine-HBr (SigmaP-7890, average chain length, 100) was reacted with 85 mg ofa-D-galactopyranosyl phenyl-isothiocyanate (Sigma G-3266). The solutionwas adjusted to pH 9 by the addition of 1/10 volume of 1M sodiumcarbonate pH 9. The tube was shielded from light by aluminum foil andmixed for 16 hours at room temperature, then dialyzed using Spectra-Pordialysis tubing (3500 M.W. cutoff) against 500 ml of 5 mM NaCl for 2days with frequent changes of buffer (4 changes/day). The reaction isstoichiometric and resulted in the galactosylation of 0.8 to 1% of theNH₃ groups present in the solution.

Basic protocol for the condensation of DNA. Plasmid DNA was preparedusing standard techniques. The DNA was resuspended in 10 mM Tris-HCl, pH8.0, containing 1 mM EDTA and the concentration of the DNA determinedspectrophotometrically. The DNA preparation was treated twice with RNAseA+T1. This step ensures that RNA is not present in the solution (RNAinhibits the condensation of DNA by poly-L-lysine). A solutioncontaining a high concentration of DNA (1.5-2 mg/ml) was used in furthersteps. An example of a typical protocol for DNA condensation isdescribed as follows:

a)300 mg of DNA in 200 ml of 0.75M NaCl (added from 5M NaCl solution) isvortexed at medium speed, using a VIBRAX apparatus (IKA-VIBRAX-VXR).This step is necessary to increase the effective length of the DNApolymer in high salt solutions, thus achieving efficient binding of thepoly-L-lysine moiety to the DNA backbone.

b)120 mg of poly-L-lysine or galactosylated poly-L-lysine (average chainlength 100) in 200 ml of 0.75M NaCl (added from a 5M NaCl solution) isadded dropwise over a period of 30 minutes to 1 hour in 5 μl aliquots.This amount translates into a molar ratio of 1 DNA PO₄ ⁻ group to 1carrier NH₃ ⁺ group.

c)The solution becomes turbid at the end of the process. Three μlaliquots of 5M NaCl are added dropwise to the vortexing solution untilturbidity disappears as monitored by eye. This process is slow, allowing60 seconds between the addition of each new aliquot of 5M NaCl. Then thesolution is subjected to circular dichroism (CD) spectroscopicmonitoring. The solutions of DNA/poly-L-lysine complexes were alsoanalyzed using a JEOL-100C electron microscope. The condensation processis complete when the diagnostic spectrum of the DNA complex is observedand is further established by EM. For subsequent preparations of DNAcomplex consisting in the same plasmid DNA at the same concentration ofnucleotide, the protocol can be followed without monitoring with CD.When using different concentration of DNA or a different plasmid the CDmonitoring should be repeated.

Animals

Six adult male Watanabe rabbits (2.8-3.2 Kg of bodyweight) were used inthese studies. These animals have been purchased from an establishedcolony at the National Institutes of Health. In order to introduce theDNA complex into the animal, we perform a single injection of 3-10 ml ofthe DNA-complex solution (.sup.˜ 400-900 mM NaCl) into the marginal earvein of the rabbit. Approximately 1.5 ml of blood was drawn from the earartery at 4 p.m. The determination of the concentration of serumcholesterol was performed in the Clinical Laboratory of UniversityHospitals of Cleveland from 300 μl of serum. At different time pointsfollowing the introduction of the DNA complex, a rabbit was subjected toa liver biopsy. Total DNA was isolated from the hepatic sample andsubjected to PCR amplification in order to detect the presence of thetransferred DNA. Rabbit #774 was treated with lovastatin (Mevacor, Merckand Dohme) orally at a dose of 10 mg per day.

Polymerase chain reaction (PCR) amplification

In order to detect the presence of the transferred DNA in the liver ofthe treated animal, total DNA was isolated from the hepatic sampleobtained upon biopsy. In the case of rabbit #737, the DNA of interestwas then amplified by PCR using an upstream primer corresponding topositions 32-50 in exon 1 of the 5' UTR of the PEPCK gene and adownstream primer complementary to nucleotides 589-607 of the human LDLreceptor cDNA. The amplified fragment corresponds to a 1100 bp band uponhybridization with a 700 bp fragment corresponding to the 5' end of thehuman LDL receptor cDNA labeled with 32P-dCTP. Appropriate primerscorresponding to the chimeric CMV-hLDL receptor gene will be used forthe PCR amplification of the transferred plasmid from liver tissueobtained from rabbit #774.

ELISA

Aliquots of 75 μl corresponding to 1 μg of DNA of either newly preparedgalactosylated-poly-L-lysine/DNA complex, plasmid DNA orgalactosylated-poly-L-lysine were incubated overnight at 4° C. to coateach well of a 96 well microtiter plate. The next day the wells werewashed 3 times with phosphate-buffered saline (PBS), then blocked for 2hours at 37° C. with 5% bovine serum albumin (BSA) in PBS and washed 3times with the washing buffer containing lo BSA and 0.5% Tween-20 inPBS. Seventy-five μl of serum from rabbit #774 obtained at differenttime points before and after the repeated administration of the DNAcomplex at dilutions of 1:3 and 1:30 were added to the wells andincubated for 90 minutes at 37° C. The wells were then washed withwashing buffer and incubated with the secondary antibody at 1:3000dilution. The secondary antibody consists of a mouse monoclonal antibodyagainst rabbit immunoglobulins conjugated to alkaline phosphatase(Sigma). After a final wash with washing buffer, the pNPP substrate at 1mg/ml in glycine buffer was added to the wells to develop the reactionand spectrophotometric readings at 410 nm were taken in a Dynatechautomated ELISA reader. Values taken at 120 minutes were chosen forcomparison.

RESULTS

1. Rabbit #676: injection of the poly-L-lysine/DNA complex containing 3mg of the chimeric PCK-hLDLR gene

In a first set of experiments,.we condensed 3 mg and 9 mg of pPCK-hLDLRwith galactosylated poly-L-lysine using the techinque developed in ourlaboratory and we injected them into the peripheral circulation ofWatanabe rabbits.

The promoter from the gene for the cytosolic form of thephosphoenolpyruvate carboxykinase (PEPCK) from the rat has beencharacterized in detail. This promoter was used in these experimentsbecause it is expressed at a high level in the liver and its expressioncan be controlled by diet and hormones. Starvation and a high protein,carbohydrate-free diet stimulate PEPCK gene transcription while a highcarbohydrate diet reduces transcription from the PEPCK promoter. Inaddition, cAMP and glucocorticoids induce, and insulin inhibits,expression of the PEPCK gene in the liver. The PEPCK promoter is thussuitable for the regulation of a linked structural gene introduced intothe liver and was used in our first experiments for the hepaticexpression of LDL receptor.

In our first approach we have injected the poly-L-lysine/DNA complexcontaining 3 mg of DNA. This basic dose of DNA was decided based onprevious experiments performed in rats. As shown in FIG. 13, theadministration of a DNA complex solution containing 3 mg of thepPCK-hLDLR plasmid in a relaxed state to rabbit #676 did not result in asignificant decrease in total serum cholesterol levels. A secondinjection of DNA complexes appropriately condensed containing 3 mg ofthe same DNA caused a 20% reduction of the levels of cholesterol in theblood. Four weeks after this second administration, cholesterol returnedto approximately pre-treatment levels, reaching a peak at about 35 days.

A 20% decrease in total serum cholesterol levels resulting from theexpression of the PCK-hLDL receptor gene will likely be helpful but willnot totally alleviate the disorder in FH patients. The number ofpoly-L-lysine/DNA complexes corresponding to 3 mg of DNA that we haveintroduced into the animal in our first approximation to theseexperiments accounts for 0.01% of the total number of asialoglycoproteinreceptors in the liver. Consequently, a linear correlation betweenincreasing concentration of.DNA complexes and expression of the PCK-hLDLreceptor gene is to be expected.

2. Rabbit #737: injection of the poly-L-lysine/DNA complex containing 9mg of the chimeric PCK-hLDLR gene

In our second experiment, 9 mg of the PCK-hLDLR gene appropriatelycondensed with galactosylated poly-L-lysine were administered to rabbit#737. As shown in FIG. 14, the treatment resulted in a 38% reduction oftotal serum cholesterol levels which lasted for about 5 weeks. Thus, a3-fold increase in the dose of DNA complex resulted in a 2-foldreduction in total serum cholesterol levels.

3. Rabbit #16: injection of the DNA complex containing 3 mg of theCMV-hLDLR gene

The promoter for the cytosolic form of the PEPCK gene has the advantageof driving expression in the liver almost specifically and in aregulated fashion. Although they are neither physiologic nor regulated,viral promoters confer high levels of expression to linked structuralgenes. The chimeric CMV promoter/enhancer has been used with success forgene therapy in WHHL rabbits using adenoviruses for gene transfer.Recently, Kozarsky et al have reported that the CMV promoter/enhancerand the chimeric β-actin/CMV promoter were the promoters of choice inorder to obtain highest expression of the human LDL receptor genetransferred to WHHL rabbits using adenoviral infection. Based on theseobservations, we injected the chimeric CMV-hLDLR gene in order toincrease the level of expression of the human LDL receptor gene in theliver of WHHL rabbits.

The administration of a DNA complex solution containing 3 mg of thechimeric CMV-hLDL receptor gene to rabbit #16 resulted in a maximalreduction of 30% in total serum cholesterol levels (FIG. 15). Elevenweeks after the injection cholesterol levels are still 20% below thoseobserved before the treatment.

4. Rabbit #775: repeated administration of the DNA complex containing 3mg of pCMV-hLDLR

Three mg of pCMV-hLDLR contained in a DNA complex solution were injectedinto rabbit #775, causing a maximal 24% reduction in the concentrationof cholesterol in the blood 3weeks after the treatment (FIG. 16A).

The life-span of hepatocytes is reported to be about 108-150 days, sothat the persistence of the introduced DNA is limited. Furthermore, alarger therapeutic effect may be of interest after a single injection ofthe DNA complex. Thus, it may be necessary to inject a patient multipletimes to ensure the appropriate level of LDL receptor in the liver. Wetested the effect of injecting the DNA complex several times into thesame animal. Rabbit #775 has been reinjected twice with 3 mg of thepCMV-hLDLR DNA complex being each injection spaced by 3 weeks. Therepeated administration of the complex did not result in a furthersignificant reduction in total serum cholesterol levels.

5. Rabbit #774: repeated administration of the DNA complex containing 3mg of pCMV-hLDLR

Rabbit #774 was injected with 3 mg of the pCMV-hLDLR complex. Weobserved a 360 decrease in the cholesterol levels in the blood (FIG.16B). To date four reinjections once every 2 weeks have been performedwith the same amount of DNA complex. Two of them have resulted in aminimal effect while the other two in a null reduction of total serumcholesterol levels. However, after five administrations of the DNAcomplex solution containing 3 mg of pCMV-hLDLR, the concentration ofcholesterol has dropped about 48% with respect to pre-treatment levels.

6. Administration of lovastatin to rabbit #774: inhibition of theendogenous synthesis of cholesterol

As described in the introduction, there is a pathway for cholesterolsynthesis inside the cell. A failure in repressing this metabolicpathway even when the hepatocyte is supplied with cholesterol throughthe uptake by the human LDL receptor could possibly inhibit furtherclearance of cholesterol. Lovastatin is a known inhibitor of HMG CoAreductase, the rate-limiting enzyme in the synthesis of cholesterol.Thus, the treatment with this drug of a rabbit that has been injectedrepeated times with the DNA complex should indicate if cholesterolsynthesis was the limiting factor for a further reduction of total serumcholesterol levels. Rabbit #774 has been treated with 10 mg oflovastatin per day for 10 weeks. A further 20% reduction in the levelsof cholesterol has been observed. The inhibition of the endogenouspathway for is cholesterol synthesis has thus brought the cholesterolconcentration of rabbit #774 to 40% of that prior the first genetransfer (FIG. 16B).

7. Injection of the DNA complex containing an irrelevant DNA

In order to control for a possible artifactual reduction in total serumcholesterol levels by injecting rabbits with the galactosylatedpoly-L-lysine/DNA complexes in a solution with high NaCl concentration(.sup.˜ 900 mM), we have administered a DNA complex solution containingan irrelevant DNA such as the luciferase gene into rabbit #775. FIG. 17shows that the injection results in a non-significant (≦12%) andtransient (≦5 days) reduction in the serum cholesterol concentration. Inaddition, we have also injected inappropriately condensed DNA complexesencoding the PCK-hLDLR gene. They result in a null or minimal andtransient decrease in the cholesterol levels in the blood as well. Thus,we have confirmed that the reduction in total serum cholesterol levelsafter the injection of appropriately condensed DNA particles encodingthe human LDL receptor gene are not a result of either the high NaClconcentration of the solution or the presence of galactosylatedpoly-L-lysine/DNA particles.

8. Detection of the transferred DNA in the liver of rabbit #774

The DNA complex used in this project is targeted to the hepaticasialoglycoprotein receptor using galactose as a ligand. It is knownthat macrophages have a similar receptor which is able to cleargalactosylated particles larger than 15 nm from the bloodstream.

In order to prove that the human LDL receptor DNA was delivered to thehepatocytes, we performed a liver biopsy in rabbit #737 60 days afterthe injection of 3 mg of the PEPCK-hLDL receptor gene. Total DNA wasisolated and subjected to PCR amplification with the primers describedabove, together with total DNA from the liver of a non-injected rabbit.The expected band of 1,100 bp was detected in the lane corresponding tothe treated rabbit but not in the non-treated animal.

9. Evaluation of the immune response of rabbit #774 after the repeatedadministration of the poly-L-lysine/DNA complex

In the field of gene therapy, immunogenicity of the delivery vehicle isoften a concern. While retroviral vectors can escape detection by theimmune system, it has been reported that adenoviral vectors do not. Thesuccess of a second administration of adenoviral particles for thetransfer into Watanabe rabbits of the human LDL receptor gene wasblocked by the onset of an immune response against the viral proteins(REF Kozarsky).

The system for receptor-mediated gene transfer has not been studied indepth in regard of its immunogenicity. It has been reported that afterthe repeated administration of anasialoorosomucoid-poly-L-lysine/DNAcomplexintomice, neutralizingantibodies against the asialoorosomucoid and poly-L-lysine components ofthe complex but not against the DNA can be detected at a dilution 1:1000(REF). Ferkol et al also reported the detection of circulatingantibodies at a 1:2000 dilution against the Fab fragment-poly-L-lysinebut not the DNA moiety of a complex upon repeated administration intomice.

We thus needed to test if the use of galactosylated-poly-L-lysine forthe condensation of DNA was immunogenic as well. For this purpose, thepresence of antibodies against the galactosylated-poly-L-lysine-DNAcomplex was evaluated in sera obtained from rabbit #774 at differenttime points before and after the repeated administration of the complex.In a first experiment, the DNA complex solution containing 1 μg of DNAwas adsorbed to the wells of a microtiter plate and then incubated withsera at dilutions 1:3, 1:30 and 1:300. Bound antibodies were detectedwith an anti-rabbit secondary antibody conjugated with alkalinephosphatase. There is an increase of antibodies in the serum of rabbit#774 upon repeated administration of the DNA complex. In fact, theystart to be detectable after the third injection of the DNA complex butnot after the first or the second. In addition, it has to be emphasizedthat only at dilutions 1:3 and 1:30 could a response be detected.

A second experiment was performed in order to establish which moiety ofthe DNA complex is responsible for inducing the weak though clear immuneresponse. We then adsorbed to the microtiter plate wells either 1 μg ofDNA, freshly prepared DNA complex containing 1 μg of DNA or thecorresponding amount of galactosylated-poly-L-lysine. The results showthat the galactosylated-poly-L-lysine moiety accounts almost entirelyfor the induction of an immune response against the complex in Watanaberabbits.

DISCUSSION

The data presented here strongly suggest that the method has been ableto at least partially correct hyperlipidemia in WHHL rabbits.

FIGS. 13-16 clearly show that a single injection of the DNA complexcontaining the human LDL receptor gene results in a significant decreaseof total serum cholesterol levels in WHHL rabbits. This reduction rangesfrom 20% in rabbit #676 to 38% in rabbit #737. In contrast, we show thatthe administration of a non-relevant plasmid DNA such aspSV40-luciferase (FIG. 17) or of a human LDL receptor-encoding plasmidthat is not appropriately condensed (FIG. 17) results in a null ornon-significant decrease in serum cholesterol.

We have used two different promoter regions for the regulation ofexpression of the human LDL receptor gene. It is tentatively suggestedthat the CMV regulatory region confers higher levels of expression inthe liver of rabbits than the promoter for the cytosolic form of the ratPEPCK gene. This observation may not be correct for every species. PEPCKactivity in the liver of rabbits is characterized by being only 10'dueto the cytosolic isozyme. In addition, stimulation of the cytosolic generesults in only a 2-fold induction of activity. Thus, the PEPCK promotermay not be the best choice for this species. But the use cf aphysiologic and tightly regulated promoter as the one for the PEPCK genemay well be the one of choice over a strong but viral promoter as theCMV in other species or for the treatment of other genetic diseases.

In order to determine the time-course of the therapeutic effect rabbits#676, #737 and #16 were subjected to a single injection of the DNAcomplex containing the human LDL receptor gene. The reduction in thelevels of cholesterol in the blood persisted for 4 weeks in rabbit #676and for 5 weeks in rabbit #737. Based on previous experiments performedin rats where the expression of the transfected pPEPCK-human Factor IXgene was shown for up to 140 days, we were expecting a longer durationof the effect. Different factors can explain this premature terminationof the corrective effect of hyperlipidemia. It is well known thatrabbits are highly immunogenic and that rats are not. The synthesis inthe WHHL rabbits of a human protein after the introduction of the humanLDL receptor gene could possibly trigger an immune response against theforeign protein, although there is an 80% homology between both speciesat the protein level. In addition, hepatocytes seem to have a limitedlife-span. Some studies in the rat indicate that the life-span ofhepatic cells is 108-150 days. Based on this observation, 40% of theincrease in cholesterol levels 5 weeks after the introduction of the DNAcomplex could result from the physiological turnover of liver cells.However, this fact cannot account for 100% of the increase. In addition,it would contradict with the long-term expression observed in ratsinjected with pPEPCK-human FIX. Another possible explanation for thepremature termination in the therapeutic effects resulting from theexpression of the human LDL receptor gene would be inactivation ordegradation of the transferred DNA.

The theoretical number of poly-L-lysine-DNA complexes that can be formedwith 3 mg of DNA accounts for 0.01% of the total number ofasialoglycoprotein receptors in the liver. Consequently, we would expectthat an increase in the dose of DNA complex results in an enhancedtherapeutic effect. To study the dose-response relationship, we haveinjected rabbit #676 with 3 mg of pPCK-hLDLR and rabbit #737 with 9 mgof the same DNA. As shown in FIGS. 13 and 14, a 3-fold increase in thedose of DNA complex results in a 2-fold higher reduction in cholesterollevels. Although these data do not establish linear correlation, anincrease in the dose clearly results in an enhanced response.

If we consider the poly-L-lysine/DNA complex as a potential drug, it isdesirable to be able to repeatedly administer it to the same animal. Forthis reason, rabbit #774 has been subjected to repeated administrationof 3 mg of the CMV-hLD>R DNA once every 2 weeks. After an initialdecrease of 36% in serum cholesterol levels following the firstinjection, the effect of the repeated administration of the DNA complexhas not been consistent. Rabbit #775 has been treated 3 times with 3 mgof the CMV-hLDLR DNA. Again, after an initial 24% reduction in thecholesterol levels, the second and third treatments have not resulted ina clear effect. We can find three possible explanations for theseresults. First, that the DNA complexes were not appropriately condensed.DNA upon condensation with poly-L-lysine can result in three differentstructures: aggregated (condensed particles out of solution), tightlycondensed and relaxed. Only DNA tightly condensed into small particlesis effective in delivering genes in vivo. Second, that the rabbits areproducing neutralizing antibodies against the vehicle. We have somepreliminary data regarding the immune response of rabbit #774 againstthe poly-L-lysine-DNA complex. Third, further clearance of cholesterolfrom the blood is limited by an impairment in the endogenous metabolismof cholesterol in the hepatocyte of the mutant Watanabe rabbit. In orderto test this last hypothesis, rabbit #774 was treated with lovastatin(10 mg/day), a known inhibitor of HMG CoA reductase, for 10 weeks. Theobservation of a further 20% reduction in the cholesterol concentrationsuggests that the inhibition of cholesterol synthesis in the hepatocyteis not complete even when the cell is supplied with cholesterol uponuptake of LDL by the heterologous LDL receptor.

Preliminary results regarding the immunogenicity of thegalactosylated-poly-L-lysine/DNA complex indicate that the repeatedadministration triggers the onset of an immune response in the Watanaberabbit. They also show that circulating antibodies can recognize thegalactosylated-poly-L-lysine but not the DNA moiety. These results agreewith previous reports regarding the immunogenicity of anasialoorosomucoid-poly-L-lysine/DNA complex and of anFab-poly-L-lysine/DNA complex. Though it is clear that the complexdesigned in our laboratory can in fact elicit an immune response uponrepeated administration in the same animal, it has to be noticed that wecould only detect circulating antibodies at much lower dilutions (1:3and 1:30 as compared to 1:1000 and 1:2000 in their case). Thisobservation might be indicative of its better ability to escapedetection by the immune system. Nevertheless, serum from more animalssubjected to repeated administration of the DNA complex need to betested for the presence of neutralizing antibodies against the complexin order to conclude that immunogenicity is responsible for the failureof repeated injections in further lowering the cholesterol levels in theWatanabe rabbits.

EXAMPLE 5

DIRECT INJECTION OF COMPLEXED VS NAKED DNA INTO MUSCLE

METHODS

Three rats per experimental set were used in the experiments involvingdirect tissue injection of the DNA complex. One hundred micrograms ofnaked- DNA containing the SV40-luciferase gene was injected into theliver and abdominal muscle of one of the animals. The same amount of theSV40-luciferase plasmid was complexed to poly-L-lysine and condensed asdescribed above and injected as well into the liver and abdominal muscleof the other two animals. The rats were sacrificed 48 hourspost-injection. A piece of liver and abdominal muscle were obtained forthe measurement of luciferase activity.

RESULTS

Evaluation of direct injections of the DNA complex into the liver andmuscle of rats. The successful transfer of naked DNA into muscle cellsof mice by direct injection has been reported. Prolonged and high levelsof expression of a chimeric gene containing the Roux sarcoma virus (RSV)regulatory region linked to the luciferase cDNA were observed in theexperiments. We have investigated the advantages of using DNA complexedto poly-L-lysine and condensed over using free DNA, when DNA has to betransferred into the liver or muscle by direct injection. Three ratshave been used for these experiments. One hundred micrograms of nakedDNA encoding SV40-luciferase were injected into the liver and abdominalmuscle of one of the animals. The same amount of the pSV40-luciferaseplasmid complexed to poly-L-lysine and condensed as described above wasinjected as well into the liver and abdominal muscle of the other twoanimals. Rats were sacrificed 48 hours post-injection. A piece of liverand abdominal muscle were homogenized in lysis buffer and cell lysateswere analyzed for luciferase activity. All luciferase measurements wereperformed in triplicate, expressed as an average of the values andstandardized for total protein. FIG. 9 shows the integrated luciferaseunits per mg of protein in the two different sets of animals. Theefficiency of transfection of DNA complexed to poly-L-lysine andcondensed seems to be slightly higher when injected into the liver.However, it appears to result in a much higher efficiency whenintroduced into muscle tissue. We observe a 20-fold higher luciferaseactivity in the sample of muscle injected with the condensed DNAcompared to the one injected with naked DNA. We think that highlycondensed and packaged DNA may be protected against nucleases and may bemore stable. In addition, poly-L-lysines may increase the efficiency ofnuclear transport once inside the cell. First, the small size of thecomplex may allow its passage through nuclear pores and second, stringsof positively charged aminoacids as lysine and arginine are known to benuclear localization signals (NLS) in various nuclear proteins.Regarding the differences found between the response in the liver and inthe muscle, it is most probable that the characteristic interconnectedstructure of skeletal muscle cells makes them a better target for thepassive diffusion of DNA from cell to cell. This would easily allow thedistribution of the DNA complex along the muscle tissue and itstransport to the nuclei.

EXAMPLE 6

DIRECT INJECTION OF NAKED VS CONDENSED DNA INTO THE BRAIN: GENE TRANSFEROF RETINAL GANGLION CELLS IN VIVO

INTRODUCTION

Insertion of foreign DNA into adult neurons has potentials for the studyof normal neuronal physiology and for the treatment of neural diseases.Gene transfer in neurons has been achieved using viral vectors, howeverit requires sophisticated methodologies and usually cells transfectedcan not be restricted to any particular type of neuron.

Axonal Retrograde transport is a continuous physiological process thathas been found to transport a large var.sup.˜ ety of different types ofmolecules. Many molecules are known to be incorporated into the axonlumen through endocytosis, whether they are adsorbed or fluid-paseparticles. in the situation where axons have been severed, it ispostulated that soluble particles from the extracellular space candiffuse into the axon and move towards the soma.

In the present experiments we tested whether plasmid DNA naked orcondensed into a compact spheroid, applied to the cut end of retinalganglion cell axons in the optic nerve or to the tectum of the brain istransported back to the soma and expressed into protein.

METHODS

Three plasmids under the control of one of three promoters which areeffective in a wide variety of eukariotic cell types were used:RSV-lacZ, CMV-lacZ and SV40-luc. They were prepared at differentconcentrations ranging from 1 to 20 μg/μl. pCMV-lacZ and pSV40-luc werecomplexed with poly-L-lysine (1:1) by Jose Carlos Perales (PNAS, 1994).

Assessment of retrograde transport of the plasmid complex to the retinalganglion cell somas was done using epifluorescence microscopyFITC-poly-L-lysine was used to form complexes with pCMV-lacz. To assessthe retrograde transport of pure plasmid, pRSV-lacZ was digested in onesite using Hind III. Biotin-dUTP was then linked to the 3'-OH ends ofpRSV-lacZ by reaction with Terminal dexynucleotidyl Transferase. Plasmidwas then precipitated and washed from free biotin-dtyrp and resuspendedat 2 μg/μl.

Adult Wistar rats were anesthetized and their optic nerves were exposed.1.5 μl of the plasmid solution (different concentrations and plasmids)was applied covering the Optic Nerve. Optic nerve axons were then cutavoiding the retinal blood supply. Another 1.5 μl of the same plasmidsolution was applied in soaked gelfoam. The conjunctiva was then closed.Same procedure was done in the contralateral eye using unspecificplasmid. Animals were sacrificed 3 days later. For direct injection intothe tectal area, nimals were anesthetized and injected stereoscopicallyinto the tectal area of the brain with naked DNA or condensed DNA.

For liquid β-galactosidase assays, retinas were kept at -70° C. untilthey were cell-lysed by repeated thawing and freezing. Tissue wascentrifuged at 12000 rpm for 2 min aiid the supernatant collected andanalyzed for protein content. Volumes containing 360 μg of protein wereincubated overnight at 37° C. in buffer A containing 15 mg/mlchlorophenol red B-D-galactopyranoside (CPRG). The absorbance wasrecorded.

For luciferase assays were done in lysis supernatants of retinas addedwith luciferase assay buffer. Samples were put into a luminometer whichwas injected with D-luciferin and then registered luminiscence.

For in situ β-galactasidase assays (for pRSV-lacZ and pCMV-lacZ) retinaswere fixed in 2% formaldehyde, 0.5%; glutaraldehyde, PBS for 30 min.,washed in PBS and incubated for 6 hrs at 37° C. in 1 mg/ml X-Gal, 4 mMpotassium ferrocyanide, 4 mM potassium ferricyanide, 2 mM MgCl₂, PBS pH7.3, 0.02% Nonidet p-40, 0.01% Deoxycholate. Tissue was then rinsed aridanalyzed immediately. Counts of blue labeled cells were made to estimatethe percentage of transfected cells.

RESULTS

1) Administration of plasmid DNA to the cut end of rat optic axonsresults in its retrograde transport to the cell body. Double labeledfield (confocal microscopy) from a retina 2 days after administration ofFITC-poly-lysine/pCMV-lacZ complex at the cut end of the optic nerve andthen incubated in propidium iodide showed that FITC (green), Propidiumiodide (red) and the mixture of both nuclei double labeled (yellow),counted in randomized fields represented about 45% of the population ofretinal ganglion cells.

Microscopic fields taken at different magnifications showed blue coloredcells in the retinal ganglion cell layer following in situβ-galactosidase assay in retina. 20 μg/μl of pRSV-lacZ were administeredat cut optic nerve and comparison was made with contralateral eyetreated with pSV40-luc. Cells positive for β-galactosidase were noted tobe in the range size known only for ganglion cells in the retina. Thesecells were counted in randomized fields and were estimated to represent35% of total ganglion cells.

2) Plasmid DNA in retinal ganglion cells is expressed in a dosedependent manner and the condensed DNA is expressed at higherefficiency.

Luciferase activity in retinas from rats whose severed optic nerves wereadministered with pSV40-luc at increasing concentrations, as comparedwith retinas just axotomized, or treated with the non-specific plasmidpCMV-lacZ (1 μg/μl) showed concentration dependent increase in activityof pSV40-luc.

The results of β-galactosidase activity in retinas from rats whosesevered optic nerves were administered with pCMV-lacZ, as compared withretinas just axotomized, or treated with non-specific plasmid pSV40-luc(10 μg/μl) showed that the highest activity was registered from themaximutn concentration of pCMV-lacZ. pCMV-lacZ complexed withpoly-lysine produced higher activity in β-galactosidase thannon-specific plasmid.

3) This method can be used in the transfer of specific genes to preciseneuronal types through their projections.

4) Intratectal injections of naked and polylysine condensed plasmid DNAcan achieve high levels of expression in the cell body of the neuronover 20 days. When the DNA is not condensed with poly-L-lysine the levelof expression returns to background after 10 days post-injection (FIG.10).

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7. Carroll, D. (1972). Optical properties of deoxyribonucleicacid-polylysine complexes. Biochemistry 11:421-426.

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9. Chenge, S. M., et al., (1975). Condensed states of nucleic acids. II.Effects of molecular size, base composition, and present ofintercalating agents on the transition of DNA. Biopolvmers 14:663-677.

10. Onge, E. C., et al., (1976). Chromatin models. The ionic strengthdependence of model histone-DNA interactions: circular dichroism.

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16. Change, C., et al., (1973). Conformational studies of nucleoprotein. Circular dichroism of deoxyribonucleic acid base pairs bound bypolylysine. Biochemistry 12:3026-3032.

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                  TABLE 101    ______________________________________                                  Present            Wu et al. Wagner et al.                                  Invention*    ______________________________________     DNA! mg/ml              ˜1    ˜0.01 ˜1    PO.sub.4 /NH.sub.3 ratio              ˜100  ˜1    ˜1.5    Buffer    150 mM NaCl 10 mM Hepes Variable                          (pH 7), 150  NaCl!                          mM NaCl    Compaction              Annealing   Direct Mixing                                      Nucleation    Method    Structure of              (Psi)       (Psi) or    Unimolecular    the DNA               Unimolecular    complex    Size of the              ≈200 nm                          80 nm       ˜10 nm    complex    Diagnostic              Gel         Electron    Circular    tools     retardation microscopy  dichroism and                                      Electron                                      microscopy    Expression in              Yes         No          Yes    vivo    Length of 6 days      --          At least 140    expression                        days    ______________________________________     *Preferred embodiment

                  TABLE 102    ______________________________________    Level off Expression of the PEPCK-hFIX Gene in the    Livers of Rats Injected with the DNA Complex    Rat #     Days after injection                           Units of hFIX activity    ______________________________________    1         2            0.040    2         2            0.045    3         4            0.045    4         4            0.025    5         6            0.330    6         8            0.135    7         12           0.160    8         12           0.075    9         32           0.125    10        48           0.350    11        72           0.005    12        136          0.105    ______________________________________

                                      TABLE 103    __________________________________________________________________________    State of DNA           Naked eye    or     (or turdi-    DNA/polyca-           metry at                  Circular Electron Absorbance at    tion complex           400 nm)                  Dichroism                           Microscopy                                    260 nm    __________________________________________________________________________    Normal DNA           No     Normal DNA                           Very thin                                    This    (Not   turbidity.                  spectrum,                           (about 1 nm                                    absorbance is    complexed)           Clear  i.e., maxima                           thick or less)                                    the reference           solution.                  at 220 and 269                           and long (about                                    for the other                  nm; a minimum                           300-1,000 nm)                                    states                  at 245 nm, and                           fibers. (Fig.                  a zero-point                           1B)                  crossover at                  258 nm.    Condensed           Low    Identical to                           Individually                                    about 20-30%    Complex           turbidity.                  the spectrum                           isolated of reference    (caused by           Almost of unbound (no                           spherical or                                    absorbance    polycation)           clear  poly-L-lysine)                           toroidal           solution.                  double   structures.                  stranded DNA                           For DNA of                  in solution;                           about 5 kb, the                  positive toroids are                  maxima at 269                           about 10-20 nm                  nm and very                           in external                  little   diameter. Larger                  contribution                           DNA, will of                  from the amide                           course compact                  bond of the                           to form larger                  poly-L-lysine                           toroids                  peptide to the                           Electron dense                  spectrum at                           particles. No                  220 nm   tibers.                  (FIG. 1A)                           (FIG. 1D)    Relaxed           No     Very difficult                           Rod-like fibers                                    about 80-100%    Complex           turbidity.                  to       (usually 10-20                                    of reference    (caused by           Clear  differentiate                           times the                                    absorbance    excess salt)           solution.                  from the diameter, of a                  condensed                           naked DNA                  form. The                           fiber, i.e.,                  only     usually 10-20                  difference is                           nm thick, and                  that there is                           longer than 60                  some     nm) of DNA and                  contribution                           branched                  from the amide                           toroidal                  bond of the                           structures of                  poly-L-lysine                           increased size                  peptide to the                           (FIG. 1F)                  spectrum at                  220 nm                  (FIG. 1A)    Precipitated           DNA fibers                  Flat spectrum.                           Complex of                                    about 1% of    Complex           in     (FIG. 1I)                           macroscopic                                    reference    (caused by           solution.       (micrometer                                    absorbance    polycation             range) DNA    if                     fibers.    insufficient    salt)    Unimolecular           Highly Characteristic                           Unimolecular                                    about 10-20%    Aggregated           variable                  red-shift and                           toroidal of reference    Complex           from fine                  positive structures                                    absorbance           particulate                  ellipticity in                           clumping           to highly                  the 300-320 nm                           together to           turbid band     form random                           networks of                           heterogeneous                           size and shape    Multimolec-           Clear  Characteristic                           Isolated,                                    about 100% of    ular          inversion in                           multimolecular                                    reference    Aggregated    the spectrum                           Toroidal absorbance    Complex       maxima at 269                           structures of    (caused by    nm to the                           variable size    polycation    negative.                           depending on    if            Clear    the number of    insufficient  contribution                           DNA molecules    salt).sup.1   from the amide                           condensed                  bond of the                           together. The                  poly-L-lysine                           size is usually                  peptide to the                           approximately 10                  spectrum at                           to 70 times                  220 mn. (FIG.                           that of the                  1H)      unimolecular                           toroids (see                           Wagner et al.                           and Shapiro et                           al.) (FIG. 1G)    __________________________________________________________________________     .sup.1 The DNA will aggregate into multimolecular complexes when the     concentration of polyL-lysine is increased suddenly in the DNA solution     (i.e. by adding polyL-lysine very rapidly to the vortexing solution of     DNA) or the direct mixing of DNA and polyL-lysine as in the method of     Shapiro also used by Wagner et al. Aggregation into multimolecular     complexes will be also the result of annealing both components     (polyL-lysine and DNA) in a gradient of decreasing NaCl concentration     (i.e. the method of Wu and Wu).

                  TABLE 104    ______________________________________          DNA          (%          super-   Initial Final  DNA! Physical                                               Acti-    Lys#  coiled)   NaCl!   NaCl!                                 (mg/ml)                                       State** vity†    ______________________________________    15*   CMV-     151.6   200   0.2   CD: ND  +          βGal                    EL: ND          (50)                         Turbidity:                                       None    20*   MT-hGH   0       267   0.85  CD: ND  -          (100)                        EL:                                       Relaxed                                       Turbidity:                                       None    27*   PEPCK-   178     439   1     CD: ND  +++          hLDLR                        EL:          (100)                        Condensed                                       Turbidity:                                       Low    56    RS-Tr    803     1000  0.24  CD: ND  ND          (50)                         EL: ND                                       Turbidity:                                       None    56    CMV-     250     746   0.2   CD: ND  ND          βGal                    EL: ND          (50)                         Turbidity:                                       Low    56*   PEPCK-   800     933   0.35  CD: ND  +++          hFIX                         EL:          (50)                         Condensed                                       Turbidity:                                       Low    56*   PEPCK-   636     970   0.6   CD: ND  +++          hFIX                         EL: ND          (50)                         Turbidity:                                       Low    109*  CMV-     500     909   0.2   CD: +   +++          βGal                    EL: ND          (50)                         Turbidity:                                       Low    109*  CMV-     689     1000  0.39  CD: ND  ND          βGal                    EL: ND          (50)                         Turbidity:                                       None    109*  CMV-     616     1036  0.95  CD: ND  +++          βGal                    EL: ND          (50)                         Turbidity:                                       Low    109*  CMV-     735     941   0.39  CD: ND  +++          βGal                    EL: ND          (50)                         Turbidity:                                       Low    109*  CMV-     500     1031  0.7   CD: +   ND          βGal                    EL: ND          (50)                         Turbidity:                                       Low    109   PEPCK-   617     1004  0.3   CD: ND  -          βGal                    EL: ND          (50)                         Turbidity:                                       None    109*  PEPCK-   1085    1174  0.88  CD: ND  +++          βGal                    EL: ND          (50)                         Turbidity:                                       Low    109*  PEPCK-   630     1063  0.8   CD: +   +++          hFIX                         EL:          (50)                         Condensed                                       Turbidity:                                       Low    109   PEPCK-   636     970   0.26  CD: ND  ND          hFIX                         EL: ND          (50)                         Turbidity:                                       None    109   PEPCK-   750     1120  0.8   CD: ND  ++          hFIX                         EL:          (50)                         Relaxed                                       Turbidity:                                       None    109*  PEPCK-   812     1098  0.7   CD: ND  +++          hFIX                         EL:          (50)                         Condensed                                       Turbidity:                                       Low    109   PEPCK-   812     1127  0.69  CD: ND  ++          hFIX                         EL:          (50)                         Relaxed                                       Turbidity:                                       None    109*  SV40-    1091    1144  0.9   CD: ND  +++          luc                          EL:          (80)                         Condensed                                       Turbidity:                                       Low    109*  SV40-    1091    1144  0.9   CD: ND  +++          luc                          EL:          (80)                         Condensed                                       Turbidity:                                       Low    109*  SV40-    961     1140  0.88  CD: ND  +++          luc                          EL: ND          (80)                         Turbidity:                                       Low    109*  SV40-    1091    1144  0.8   CD: ND  +++          luc                          EL: ND          (80)                         Turbidity:                                       Low    109   SV40-    666     1000  0.19  CD: +   ND          luc                          EL:          (80)                         Relaxed                                       Turbidity:                                       None    109*  SV40-    961     1121  0.8   CD: ND  +++          luc                          EL: ND          (80)                         Turbidity:                                       None    109*  SV40-    735     972   0.55  CD: ND  +++          luc                          EL: ND          (80)                         Turbidity:                                       Low    109*  Salmon   900     1231  1     CD: ND  ND          sperm                        EL: ND          DNA (0)                      Turbidity:                                       None    109   PEPCK-   774     948   0.9   CD: ND  ND          OTC                          EL: ND          (50)                         Turbidity:                                       Low    123   SV40-    719     1044  0.95  CD: ND  -          luc                          EL:          (100)                        Relaxed                                       Turbidity:                                       None    123   SV40-    905     1086  1     CD: ND  -          luc                          EL:          (100)                        Relaxed                                       Turbidity:                                       None    123   SV40-    689     1019  0.95  CD: ND  -          luc                          EL: ND          (100)                        Turbidity:                                       None    123   SV40-    783     978   0.5   CD: ND  -          luc                          EL: ND          (100)                        Turbidity:                                       None    123   SV40-    905     1149  0.57  CD: ND  -          luc                          EL:          (100)                        Relaxed                                       Turbidity:                                       None    123*  CMV-     825     1020  0.76  CD: ND  ND          βGal                    EL: ND          (ND)                         Turbidity:                                       None    150*  CMV-     886     1077  0.5   CD: ND  +++          βGal                    EL:          (ND)                         Condensed                                       Turbidity:                                       Low    150*  SV40-    800     972   0.36  CD: ND  +++          luc                          EL: ND          (80)                         Turbidity:                                       Low    150   SV40-    821     868   0.3   CD: Psi -          luc                          DNA          (80)                         EL:                                       Aggregated                                       Turbidity:                                       High    150*  SV40-    821     968   0.3   CD: +   +++          luc                          EL:          (80)                         Condensed                                       Turbidity:                                       Low    150   SV40-    821     1071  0.3   CD: +   -          luc                          EL:          (80)                         Relaxed                                       Turbidity:                                       None    240*  SV40-    711     1125  1     CD: ND  +++          luc                          EL:          (80)                         Condensed                                       Turbidity:                                       Low    240   SV40-    711     1162  1     CD: ND  +          luc                          EL:          (80)                         Relaxed                                       Turbidity:                                       Low    240   SV40-    711     1280  1     CD: ND  -          luc                          EL:          (80)                         Relaxed                                       Turbidity:                                       None    240   SV40-    800     1007  1     CD: ND  -          luc                          EL:          (80)                         Aggregated                                       Turbidity:                                       High    240   T7-T7    708     1187  0.9   CD: +   ND          (90)                         EL:                                       Condensed                                       Turbidity:                                       Low    240   T7-T7    708     1250  0.9   CD: +   -          (90)                         EL:                                       Relaxed                                       Turbidity:                                       None    240   PEPCK-   642     947   0.73  CD: Psi -          hLDLR                        DNA          (100)                        EL:                                       Aggregated                                       Turbidity:                                       None    240   PEPCK-   706     1174  0.35  CD: ND  ND          OTC                          EL: ND          (50)                         Turbidity:                                       None    240   PEPCK-   898     1153  0.64  CD: ND  ND          OTC                          EL: ND          (50)                         Turbidity:                                       None    ______________________________________     *Used in compiling Table 105.     ND = Not determined     **Physical state of the DNA complex after polycation binding.     1. When circular dichroism (CD) was determined the results are indicated     as follows: spectral changes due to the polycation condensation of DNA ar     insignificant (+); polycation condensation resulted in Psiform DNA due to     aggregation into multimolecular complexes (either rodlike or toroidal)     (Psi DNA); appearance of an aberrant spectrum associated with a highly     aggregative state (-).     2. Electron microscopic results have been indicated as follows: the     association of the polycation with the DNA results in aggregation into     complexes of increased size (>60 nm) (Aggregated); the structures     resulting from the condensation are rodlike relaxed toroids of increased     size (Relaxed); polycation binding results in proper condensation (toroid     <30 nm in diameter) (Condensed). The number of properly condensed     structures (toroids) per microscopic field has not been determined. There     is approximately 3fold variation in the number of toroids visible in the     EL with different preparations of DNA complex.     3. Turbidity measurements are based on visual inspection of the final     solution of DNA complex.     †A relative indication of the activity of the introduced gene afte     introduction of the DNA complex: hFIX (human factor IX) is measured by th     western blot hybridization or by a functional activity assay of rat plasm     samples.     βGal (galactosidase) activity is measured by in situ histochemistry     in fixed cells or tissue sections.     luc (luciferase) activity is measured using a specific enzyme activity     assay with tissue extracts.     hLDLR (human LDL receptor) activity was measured indirectly after     determination of the total serum cholesterol levels in a rabbit model for     LDL receptor deficiency.     hGH (human growth hormone) activity refers to a direct measurement of hGH     levels in the serum of animals transfected with the DNA complex. A     radioimmuno assay specific for hGH was used.     The activity is relative to all the experiment performed with the same     DNA. Not detectable activity after introduction of the DNA complex is     indicated by "-".

                                      TABLE 105    __________________________________________________________________________    Final  NaCl! = -555.75 +  DNA! mg/ml*180.91 + log (lys length)*y18.32    Regression Statistics    Multiple R0.881909585    R Square0.777764515    Adjusted R Square      0.743574441    Standard Error135.5087624    Observations16    Analysis of Variance    __________________________________________________________________________           df  Sum of Squares                       Mean Square                               F     Significance F    __________________________________________________________________________    Regression            2  835435.3166                       417717.6583                               22.748254                                     5.6792E-05    Residual           13  238714.1209                       18362.62469    Total  15  1074149.438    __________________________________________________________________________           Coefficients                  Standard Error                         t Statistic                                P-value                                     Lower 95%    __________________________________________________________________________    Intercept           -555.757861                  228.34416556                         2.433887324                                0.0279103                                     -1049.059922     DNA! mg/ml           180.9113279                  125.4285365                         1.442345841                                0.1697596                                     -90.06049864    log (lys length)           718.3211054                  117.7844848                         6.098605488                                2.037E-05                                     463.8632453    __________________________________________________________________________

                  TABLE 106    ______________________________________    Estimated and experimental size of condensed DNA    complexes                 Condensed diameter (nm ± SD)                                   Hydrated                                          Hydrated                                   model  model (X-                                   (partial                                          ray                       Electron    specific                                          diffraction    DNA       size(bp) Microscope.sup.a                                   volume).sup.b                                          density).sup.c    ______________________________________    PEPCK-hFIX              4,500    12.80 ± 1.56                                   18     22    PEPCK-hOTC              5,300    18.00 ± 1.83                                   20     23    SV40-luciferase              5,600    16.95 ± 3.50                                   20     24    PEPCK-CAT 5,800    16.30 ± 2.56                                   20     24    CMV-hLDLr 7,400    20.70 ± 2.60                                   22     26    φ29.sup.d              18,000   38.sup.c    40     47    ______________________________________     .sup.a measured diameter of at least 10 DNA complexes in a printed     photograph (× 240,000).     .sup.b calculated diameter of a unimolecular DNA complex assuming a     condensed sphere. The partial specific volume of NaDNA was deemed to be     0.5 ml/g. The contribution of galactosylated polyL-lysine at a charge     ratio of 1:1 has been added. The molecular weight of DNA was calculated     based on an average molecular weight of 6,500 dalton/10 bp. The formula     used is: DNA molecular weight (daltons)/6.023 × 10.sup.23 ×     0.5 (ml/g) = ml occupied by a molecule of DNA of X molecular weight.     Diameter obtained from the formula for the volume of a sphere.     .sup.c calculated diameter of a unimolecular DNA complex assuming a     condensed sphere. The calculation assumed a hydrated density of 1.25 ±     0.1 g/ml as determined by Xray difraction. The contribution of a     galactosylated polyL-lysine at a charge ratio of 1:1 has been added. The     molecular weight of DNA was calculated based on an average molecular     weight of 6,500 dalton/10 bp. The formula is: DNA molecular weight     (daltons)/6.023 × 10.sup.23 × 1.25 (g/ml) = ml occupied by a     molecular of DNA of X molecular weight. Diameter obtained from the formul     for the volume of a sphere.     .sup.d from the literature.     .sup.e the size to the phage prohead includes the protein outshell.

We claim:
 1. A non-naturally occurring composition comprisingunaggregated nucleic acid complexes, each complex consisting essentiallyof a single nucleic acid molecule and one or more carrier molecules,said carrier molecule having a nucleic acid binding moiety through whichit is complexed to the nucleic acid, and a target cell-binding moietythrough which it may bind to a target cell and whereby the complex maymore readily enter the target cell, wherein said complex is compacted toa diameter which is less than (a) double the theoretical minimumdiameter of a complex of said single nucleic acid molecule and asufficient number of carrier molecules to provide a charge ratio of 1:1,in the form of a condensed sphere, or (b) 30 nm, whichever is larger. 2.The composition of claim 1, wherein said complex is compacted to adiameter of less than 90 nm.
 3. The composition of claim 1, wherein thenucleic acid complex is compacted to a diameter less than 30 nm.
 4. Thecomposition of claim 1, wherein the nucleic acid complex is compacted toa diameter less than 23 nm.
 5. The composition of claim 1, wherein thenucleic acid complex is compacted to a diameter not more than 12 nm. 6.The composition of claim 1 wherein the target cell binding moiety is anantibody or a specific binding fragment of an antibody.
 7. Thecomposition of claim 1 in which the nucleic acid is 100 to 100,000bases, if single stranded, or 100 to 100,000 base pairs, if doublestranded.
 8. The composition of claim 6 in which the antibodyspecifically binds CD4 or gp120.
 9. The composition of claim 1 whereinthe target cell binding moiety is a lectin, or a carbohydrate.
 10. Thecomposition of claim 9 wherein the target cell binding moitey is acarbohydrate selected from the group consisting of galactose, lactose,mannose and mannose-6-phosphate.
 11. The composition of claim 1 whereinthe target cell binding moiety is a peptide or protein.
 12. Thecomposition of claim 11 wherein the target cell binding moiety isselected from the group consisting of insulin, epidermal growth factor,tumor necrosis factor, prolactin, chorionic gonadotropin, folliclestimulating hormone, luteinizing hormone, glucagon, lactoferrin,transferrin, apolipoprotein E, gp120 and albumin.
 13. The composition ofclaim 1 in which the nucleic acid binding moiety is an antibody or aspecific binding fragment of an antibody.
 14. The composition of claim 1in which the nucleic acid binding moiety is a polycation.
 15. Thecomposition of claim 1 in which the polycation is polylysine.
 16. Thecomposition of claim 1 in which the nucleic acid is DNA.
 17. Thecomposition of claim 1 in which the nucleic acid is RNA.
 18. Thecomposition of claim 1 in which the nucleic acid is an analogue of DNAor RNA which is more resistant to degradation in vivo.
 19. Thecomposition of claim 1 in which the nucleic acid is an analogue of DNAor RNA which more readily diffuses through cell membranes.
 20. Thecomposition of claim 1 in which the nucleic acid is an analogue of DNAin which the methylphosphonate analogue of the naturally occurringmononucleotide appears.
 21. The composition of claim 1 wherein thenucleic acid comprises an expressible gene which is functional in atarget cell to which said target cell binding moiety binds.
 22. Thecomposition of claim 1 in which the gene encodes a protein selected fromthe group consisting of enzymes which in mutated form are associatedwith specific metabolic defects, receptors, toxins, ion channelsmembrane transporters, and cytoskeletal proteins.
 23. The composition ofclaim 1 wherein the nucleic acid comprises sequences homologous togenetic material of the target cell, whereby it may be inserted into thegenome thereof by homologous recombination.
 24. The composition of claim1 in which the nucleic acid molecule is "antisense" to a target nucleicacid sequence of the target cell, or of a virus which can infect thetarget cell, whereby it may hybridize sufficiently thereto to inhibittranscription or translation of the target nucleic acid sequence. 25.The composition of claim 1 wherein the nucleic acid is encapsulated in aliposome.
 26. The composition of claim 1 wherein the nucleic acidbinding moiety is covalently linked to the target cell binding moiety.27. The composition of claim 1 wherein the nucleic acid molecule iscDNA.
 28. The composition of claim 1 wherein the complex is compacted toa diameter which is less than is less than (a) double the theoreticalminimum diameter of a complex of said single nucleic acid molecule and asufficient number of carrier molecules to provide a charge ratio of 1:1,in the form of a condensed sphere.
 29. A composition comprisingunaggregated nucleic acid complexes, each complex consisting essentiallyof a single double-stranded nucleic acid molecule and one or morecarrier molecules, said carrier molecule having a nucleic acid bindingmoiety through which it is complexed to the nucleic acid, and a targetcell-binding moiety through which it may bind to a target cell andwhereby the complex may more readily enter the target cell, wherein saidcomplex is compacted to a diameter which is less than (a) double thetheoretical minimum diameter of a complex of said single nucleic acidmolecule and a sufficient number of carrier molecules to provide acharge ratio of 1:1, in the form of a condensed sphere, or (b) 30 nm,whichever is larger.
 30. The composition of claim 29 in which thenucleic acid is DNA.
 31. The composition of claim 29, wherein thenucleic acid complex is compacted to a diameter not more than 12 nm. 32.The composition of claim 29, wherein the target cell binding moiety isan antibody or a specific binding fragment of an antibody.
 33. Thecomposition of claim 32 in which the antibody specifically binds CD4 orgp120.
 34. The composition of claim 29 wherein the target cell bindingmoiety is a lectin, or a carbohydrate.
 35. The composition of claim 34wherein the carbohydrate is selected from the group consisting ofgalactose, lactose, mannose and mannose-6-phosphate.
 36. The compositionof claim 29 wherein the target cell being moiety is selected from thegroup consisting of insulin, epidermal growth factor, tumor necrosisfactor, prolactin, chorionic gonadotropin, follicle stimulating hormone,luteinizing hormone, glucagon, lactoferrin, transferrin, apolipoproteinE, gp120 and albumin.
 37. The composition of claim 29 in which thenucleic acid binding moiety is an antibody or a specific bindingfragment of an antibody.
 38. The composition of claim 29 in which thepolycation is polylysine.
 39. The composition of claim 29 in which thenucleic acid is an analogue of DNA or RNA which is more resistant todegradation in vivo.
 40. The composition of claim 29 in which thenucleic acid is an analogue of DNA or RNA which more readily diffusesthrough cell membranes.
 41. The composition of claim 29 in which thenucleic acid is an analogue of DNA in which the methylphosphonateanalogue of the naturally occurring mononucleotide appears.
 42. Thecomposition of claim 29 in which the nucleic acid molecule is"antisense" to a target nucleic acid sequence of the target cell, or ofa virus which can infect the target cell, whereby it may hybridizesufficiently thereto to inhibit transcription or translation of thetarget nucleic acid sequence.
 43. The composition of claim 29 whereinthe nucleic acid is encapsulated in a liposome.
 44. The composition ofclaim 29, wherein the nucleic acid complex is compacted to a diameterless than 23 nm.
 45. The composition of claim 29 wherein the target cellbinding moiety is a peptide or protein.
 46. The composition of claim 29in which the nucleic acid is RNA.
 47. The composition of claim 29wherein the nucleic acid comprises an expressible gene which isfunctional in a target cell to which said target cell binding moietybinds.
 48. The composition of claim 29 in which the gene encodes aprotein selected from the group consisting of enzymes which in mutatedform are associated with specific metabolic defects, receptors, toxins,ion channels membrane transporters, and cytoskeletal proteins.
 49. Thecomposition of claim 29 wherein the nucleic acid comprises sequenceshomologous to genetic material of the target cell, whereby it may beinserted into the genome thereof by homologous recombination.
 50. Thecomposition of claim 29 in which the nucleic acid is 100 to 100,000 basepairs.
 51. The composition of claim 29, wherein said complex iscompacted to a diameter of less than 90 nm.
 52. The composition of claim29, wherein the nucleic acid complex is compacted to a diameter lessthan 30 nm.
 53. The composition of claim 29 in which the nucleic acid is100 to 100,000 base pairs.
 54. The composition of claim 29 in which thenucleic acid binding moiety is a polycation.
 55. The composition ofclaim 29 wherein the nucleic acid binding moiety is covalently linked tothe the target cell binding moiety.
 56. The composition of claim 29wherein the nucleic acid molecule is cDNA.
 57. The composition of claim29 wherein the complex is compacted to a diameter which is less than isless than (a) double the theoretical minimum diameter of a complex ofsaid single nucleic acid molecule and a sufficient number of carriermolecules to provide a charge ratio of 1:1, in the form of a condensedsphere.